Anti-CEA antibody designated 806.077, hybridoma and method of manufacture

ABSTRACT

An anti-CEA monoclonal antibody, designated 806.077, of murine origin is useful for the diagnosis and therapy of cancer. The antibody complementarity determining regions have the following sequences: heavy chain CDR1 DNYMH, CDR2 WIDPENGDTE YAPKFRG, CDR3 LIYAGYLAMD Y; and light chain CDR1 SASSSVTYMH, CDR2 STSNLAS, CDR3 QQRSTYPLT. The antibody optionally is humanized and can be in the form of a conjugate with either an enzyme, such as carboxypeptidase, or a co-stimulatory molecule such as the extracellular domain of human B7.1.

This application is a § 371 national phase filing of PCT/GB97/01165,filed Apr. 29, 1997.

The present invention relates to a novel anti-CEA monoclonal antibody(named “806.077 antibody” or “806.077 Ab” herein) useful for thediagnosis and therapy of cancer.

It is established that the transformation of normal tissue cells totumour cells is associated with a change in structure on the cellsurface. Altered cell surface structures can serve as antigens and thetumour modified structures represent a type of so-calledtumour-associated antigen (see for example Altered Glycosylation inTumour Cells, Eds. Reading, Hakamori and Marcus 1988, Arthur R. Lisspubl.). Such antigens may be exploited for example by the generation ofmonospecific antibodies using hybridoma technology as is presently wellestablished after being first described by Kohler and Milstein (Nature,256, 495-497, 1975).

One tumour-associated antigen is CEA (Carcinomembryonic Antigen) asfirst described by Gold and Freedman, J Exp Med, 121 439, 1965. Thisantigen is present on the tumour cell surface and can also bedemonstrated in blood serum.

The concept of using antibodies to target tumour associated antigens inthe treatment of cancer has been appreciated for some time (Herlynet.al. (1980) Cancer Research 40 717). Antibodies may be used to targetvarious chemical and biological agents to the tumour and such conjugateshave been particularly successful in forming the basis for many methodsof both in vitro and in vivo diagnosis. The use of immunoconjugates inthe therapy of cancer is also promising (Lord et al.(1985) Trends inBiotechnology 3, 175; Vitetta et al (1987) Science 238, 1098). Thisapproach is technically more demanding than diagnostic applications andrequires that tumour associated antigens which are targetted in suchimmunotherapeutic approaches, are highly tumour specific and notexpressed at significant levels in vital human tissues. Whilst notwishing to be bound by theoretical considerations, as well as theproperty of having specific tumour associated tissue distribution, forsome applications it is desirable that the antibody remain at the cellsurface after antigen binding rather than being quickly internalised.For example in ADEPT (antibody directed enzyme prodrug therapy, see U.S.Pat. Nos. 4,975,278 and 5,405,990) it is believed to be preferred thatthe antibody remain at the cell surface to facilitate prodrug activationby antibody-enzyme conjugate.

Antibody conjugates also have application in tumour immunotherapy. Thefollowing few paragraphs set out the scientific background for thisapplication. In order to respond to an immune stimulus, T-cells requiretwo signals. One such signal is provided by recognition of MHC displayedpeptides by the T-cell receptor (TCR). It has been demonstrated howeverthat TCR stimulation alone results in T-cell unresponsiveness or anergyand a second or co-stimulatory signal is required to stimulate specificT-cell activation and proliferation (reviewed by Schwartz R. H.J.Exp.Med., 1996, 184, 1-8). Upon receiving both signals, the resultingcytotoxic T-cells mediate the immune response by killing the targetcells. A number of potential co-stimulatory molecules have beenidentified (eg B7, ICAMs, LFA-1 and 3, CD40, CD70 and CD24, reviewed byGalea-Lauri J. et al Cancer Gene Therapy, 1996, 3, 202-213). The majorco-stimulatory function appears to be provided by the related moleculesB7.1 (also called CD80) and B7.2 (also called CD86) which can interactwith two receptors, CD28 and CTLA-4 (Hellstrom K. E. et al Immunol.Rev., 1995, 145, 123-145 and Lenschow D. J. et al Ann.Rev.Immunol.,1996, 14, 233-258). B7.1 and B7.2 are expressed on antigen presentingcells (APC) such as dendritic cells whereas CD28 and CTLA-4 are presenton T-cells. B7.2 appears to be constitutively expressed on the surfaceof APCs but after contact with a T-cell, expression of B7.1 isup-regulated. Analogously, CD28 is expressed on T-cells but afteractivation is down-regulated and replaced by CTLA-4 expression. Thestimulation of CD28 and CTLA-4 by B7.1 and B7.2 represents a complexpattern of signalling which controls not only the activation of theT-cell, but the subsequent control of proliferation to modulate theimmune-response (Greene J. et al J.Biol.Chem., 1996, 271, 26762-26771).This phenomenon may explain the sometimes conflicting data reported byworkers studying these co-stimulatory molecules.

In cancer, tumour infiltrating lymphocytes have been identified but thelack of immune-response to the tumour may be due to T-cell anergy.Tumour cells can display specific or selective antigens on their surfacebut lack B7.1/B7.2 allowing them to escape immune surveillance. Indeed,in vivo experiments have demonstrated that B7.1/B7.2 transfected tumourcells are less tumourigenic than untransfected cells from the same lineand that the transfected cells are capable of inducing protectiveimmunity against rechallenge with parental cells (Townsend S. E. andAllison J. P., 1993, Science, 259, 368-370). This demonstrates that oncestimulated, the immune response can become B7.1/B7.2 independent.Hellstrom has proposed that expression of B7.1/B7.2 in tumour cells bygene therapy has the potential to stimulate a host reponse which canreduce or eliminate the disease. Gajewski (J.Immunol., 1996, 156,465-472) and Matulonis et al (J.Immunol., 1996, 156, 1126-1131) havereported that B7.1 is superior to B7.2 in the activation of T-cells. Theuse of B7.1 in solution (as a fusion with antibody constant domains) isreported to provide only modest co-stimulation to T-cells receiving TCRstimulation via an independent source (Linsley P.S. et al J.Exp.Med.,1991, 173, 721-730).

There is a need for further and improved anti-CEA antibodies useful incancer diagnosis and therapy.

The present invention is based on the discovery of a novel anti-CEAantibody termed 806.077 antibody herein.

According to one aspect of the present invention there is provided ananti-CEA antibody comprising complementarity determining regions (CDRs)in which the CDRs have the following sequences:

a) heavy chain

CDR1 DNYMH (SEQ ID NO: 29)

CDR2 WIDPENGDTE YAPKFRG (SEQ ID NO: 31)

CDR3 LIYAGYLAMD Y(SEQ ID NO: 32);

b) light chain

CDR1 SASSSVTYMH (SEQ ID NO: 26)

CDR2 STSNLAS (SEQ ID NO: 27)

CDR3 QQRSTYPLT (SEQ ID NO: 28).

The CDRs or complementarity determining regions are those sequenceswithin the hypervariable loops of antibody variable domains which arebelieved to be critical in determining the specificity of theantigen-antibody interaction (Kabat, E. A., Lu, T. T., Reid-Miller, M.,Perry, H. M. & Gottesman, K. S. (1987). Sequences of Proteins ofImmunological Interest. 4th edition. Washington D.C.: United StatesDept. of Health and Human Services; the reader is also referred to thisreference for details of Kabat antibody residue numbering). CDRs adefined herein however include framework residues where these contributeto binding. For the 806.077 antibody the CDRs were determined byhomology with the hypervariable sequences of other murine antibodies. Inthis specification the terms “VK” and “VH” mean variable regions of thelight and heavy antibody chains respectively. Anatomy of the antibodymolecule has been reviewed by Padlan (1994) in Molecular Immunology 31,169-217.

The Light Chain CDRs are:

VK CDR1 Kabat residues 24-34 inclusive, SASSSVTYMH (SEQ ID NO: 26);

VK CDR2 Kabat residues 50-56 inclusive, STSNLAS (SEQ ID NO: 27);

VK CDR3 Kabat residues 89-97 inclusive, QQRSTYPLT (SEQ ID NO: 28);

The Heavy Chain CDRs are:

VH CDR1 Kabat residues 31-35B inclusive, DNYMH (SEQ ID NO: 29);

preferred VH CDR1 Kabat residues are no. 27-35B inclusive, FNIKDNYMH(SEQ ID NO: 30);

VH CDR2 Kabat residues 50-65 inclusive, WIDPENGDTE YAPKFRG (SEQ ID NO:31)

VH CDR3 Kabat residues 95-102 inclusive, LIYAGYLAMD Y (SEQ ID NO: 32);and

preferred VH CDR3 Kabat residues are no. 93-102 inclusive, HVLIYAGYLAMDY (SEQ ID NO: 33).

Preferably binding affinity for CEA antigen is at least 10E-5M, morepreferably binding affinity for CEA is at least 10E-6M, more preferablybinding affinity for CEA is at least 10E-7M, more preferably bindingaffinity for CEA is at least 10E-8M, more preferably binding affinityfor CEA is at least 10E-9M, more preferably binding affinity for CEA isat least 10E-10M and especially binding affinity for CEA is at least10E-11M.

The term antibody as used herein generally means an immunoglobulinmolecule (or fragment thereof or modified antibody construct such asscFv which retains specific CEA antigen binding). The CDRs areprincipally responsible for antigen binding, the non-CDR proteinsequence is normally derived from an immunoglobulin but may be derivedfrom immunoglobulin domain of a immunoglobulin super family member.

According to another aspect of the present invention there is provided aCEA antibody comprising the following, optionally humanised, structure:

a heavy chain variable region sequence (SEQ ID NO: 11)

EVQLQQSGAE LVRSGASVKL SCTASGFNIK DNYMHWVKQR 40

PEQGLEWIAW IDPENGDTEY APKFRGKATL TADSSSNTAY 80

LHLSSLTSED TAVYYCHVLI YAGYLAMDYW GQGTSVAVSS 120

and;

a light chain variable region sequence (SEQ ID NO: 9):

DIELTQSPAI MSASPGEKVT ITCSASSSVT YMHWFQQKPG 40

TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE 80

DAATYYCQQR STYPLTFGAG TKLELKRA 108;

or any one of the following constructs thereof:

F(ab′)₂; F(ab′), Fab, Fv, single chain Fv & V-min.

F(ab′)₂ fragment constructs are preferred. Any suitable antibodyfragment which retains 806.077 antibody binding characteristics iscontemplated. For example a recently described antibody fragment is“L-F(ab)₂” as described by Zapata (1995) in Protein Engineering, 8,1057-1062. Disulphide bonded Fvs are also contemplated. Optionally theantibody forms part of a conjugate as described below.

A preferred humanised antibody comprises at least one of the followingsequences:

a heavy chain variable region sequence which is VH1 (SEQ ID NO: 55);

a light chain variable region sequence which is VK4 (SEQ ID NO: 71);

a human CH1 heavy chain IgG3 constant region;

a human kappa light chain CL region; and

a human IgG3 hinge region;

optionally in the form of a F(ab′)₂ fragment.

According to another aspect of the present invention there is provided apolynucleotide sequence capable of encoding for the heavy or light chainvariable region of a CEA antibody of the invention. Preferably the heavyor light chain variable region is fused (optionally via some linkingsequence) to a gene encoding a protein effector moiety (as part of aconjugate, see text below), preferably fusion is through the antibodyheavy chain. Generally fusion can be either at the N or C terminus ofthe antibody chain. For B7 conjugates fusion at the N-terminus of theantibody chain is preferred.

CPB has an N-terminal pro domain which is believed to assist correctfolding of protein before the pro domain is removed to release activeenzyme. If proCPB is fused at its C terminus to the N terminus of anantibody chain this allows removal of pro domain (e.g. by trypsintreatment) from the N terminus of the fusion construct. Alternatively ifproCPB was attached to the C terminus of an antibody chain then theproblem arises of having to remove the pro domain from the “middle” ofthe construct without destroying the fusion protein. The solution is toco-express the pro domain separately (in trans). This has the advantage,once the cell lines have been constructed, of not requiring trypsinactivation of expressed fusion protein to remove CPB pro domain.Constructs with proCPB fused at its C terminus to the N terminus of anantibody chain have the advantage of not requiring construction ofco-expression cell lines which require high level expression of the prodomain along -with high level expression of other proteins.

In this specification conservative amino acid analogues of specificamino acid sequences are contemplated which retain the bindingproperties of the CEA antibody of the invention but differ in sequenceby one or more conservative amino acid substitutions, deletions oradditions. However the specifically listed amino acid sequences arepreferred. Typical conservative amino acid substitutions are tabulatedbelow.

Conservative Substitutions Exemplary Preferred Original SubstitutionsSubstitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) AsnAsn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Leu Norleucine Leu (L) Norleucine; Ile;Val; Met; Ile Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; IleLeu Phe (F) Leu; Val; Ile; Ala Leu Pro (P) Gly Gly Ser (S) Thr Thr Thr(T) Ser Ser Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe;Ala; Leu Norleucine

In this specification nucleic acid variations (deletions, substitutionsand additions) of specific nucleic acid sequences are contemplated whichretain which the ability to hybridise under stringent conditions to thespecific sequence in question. A hybridisation test is set out inExample 9 hereinafter. However specifically listed nucleic acidsequences are preferred. It is contemplated that peptide nucleic acidmay be an acceptable equivalent of polynucleotide sequences, at leastfor purposes that do not require translation into protein (Wittung(1994) Nature 368, 561).

According to another aspect of the present invention there is providedan antibody or antibody fragment as herein described characterised inthat it is humanised.

A humanised antibody, related fragment or antibody binding structure isa polypeptide composed largely of a structural framework of humanderived immunoglobulin sequences supporting non human derived amino acidsequences in and around the antigen binding site (complementaritydetermining regions or CDRs; this technique is known as CDR graftingwhich often involves some framework changes too, see the Examplesbelow). Appropriate methodology has been described for example in detailin WO 91/09967, EP 0328404 and Queen et al. Proc Natl Acad Sci 86,10029,Mountain and Adair (1989) Biotechnology and Genetic Engineering Reviews10, 1 (1992) although alternative methods of humanisation are alsocontemplated such as antibody veneering of surface residues (EP 519596,Merck/NIH, Padlan et al). Preferred humanised 806.077 antibodies are anyone of Examples 11-47 or 107-122. A preferred humanised heavy chainvariable region is VH1 (see Examples). A preferred light chain variableregion is VK4 optionally incorporating any of the additional changesdescribed in Examples 107-109. A preferred human heavy chain constantregion is IgG3.

Chimaeric humanised antibodies represent another aspect of theinvention. Preparation of chimaeric humanised antibody fragments ofantibody 806.077 antibody is described in Example 8 herein. Chimaericantibodies are generally constructed by combining the variable regionfrom one species with a constant region from another antibody from adifferent species.

The term “humanised” in relation to antibodies as used herein includesany method of humanisation such as for example CDR grafting or chimaericantibody preparation or any hybrid thereof such as for example a CDRgrafted heavy chain in combination with a chimaerised light chain (seeExample 110 for a suitable embodiment).

In particular, a rodent antibody on repeated in vivo administration inman either alone or as a conjugate will bring about an immune responsein the recipient against the rodent antibody; the so-called HAMAresponse (Human Anti Mouse Antibody). The HAMA response may limit theeffectiveness of the pharmaceutical if repeated dosing is required. Theimmunogenicity of the antibody may be reduced by chemical modificationof the antibody with a hydrophilic polymer such as polyethylene glycolor by using the methods of genetic engineering to make the antibodybinding structure more human like. For example, the gene sequences forthe variable domains of the rodent antibody which bind CEA can besubstituted for the variable domains of a human myeloma protein, thusproducing a recombinant chimaeric antibody. These procedures aredetailed in EP 194276, EP 0120694, EP 0125023, EP 0171496, EP 0173494and WO 86/01533. Alternatively the gene sequences of the CDRs of the CEAbinding rodent antibody may be isolated or synthesized and substitutedfor the corresponding sequence regions of a homologous human antibodygene, producing a human antibody with the specificity of the originalrodent antibody. These procedures are described in EP 023940, WO90/07861 and WO91/09967. Alternatively a large number of the surfaceresidues of the variable domain of the rodent antibody may be changed tothose residues normally found on a homologous human antibody, producinga rodent antibody which has a surface ‘veneer’ of residues and whichwill therefore be recognized as self by the human body. This approachhas been demonstrated by Padlan et.al. (1991) Mol. Immunol. 28, 489.

According to another aspect of the present invention there is provided ahost cell transformed with a polynucleotide sequence or a transgenicnon-human animal or transgenic plant developed from the host cell inwhich the polynucloetide sequence encodes at least the variable regionof the heavy chain or light chain of a CEA antibody of the invention,optionally in the form of a conjugate as described herein.

According to another aspect of the present invention there is providedhybridoma 806.077 deposited as ECACC deposit no. 96022936 and variantcell lines thereof.

Hybridoma 806.077 was deposited at the European Collection of AnimalCell Cultures (ECACC), PHLS Centre for Applied Microbiology & Research,Porton Down Salisbury, Wiltshire SP4 0JG, United Kingdom on Feb. 29,1996 under accession no. 96022936 in accordance with the BudapestTreaty.

According to another aspect of the present invention there is providedplasmid pNG3-Vkss-HuCk deposited as NCIMB deposit no.40798.

Plasmid pNG3-Vkss-HuCk was deposited at The National Collections ofIndustrial and Marine Bacteria (NCIMB), 23 St Machar Drive, Aberdeen AB21RY, Scotland, United Kingdom on Apr. 11, 1996 under deposit referencenumber NCIMB 40798 in accordance with the Budapest Treaty.

According to another aspect of the present invention there is providedplasmid pNG4-VHss-HuIgG2CH1′ deposited as NCIMB deposit no. 40797.

Plasmid pNG4-VHss-HulgG2CH1′ was deposited at The National Collectionsof Industrial and Marine Bacteria (NCIMB), 23 St Machar Drive, AberdeenAB2 1RY, Scotland, United Kingdom on Apr. 11, 1996 under depositreference number NCIMB 40797 in accordance with the Budapest Treaty.

According to another aspect of the present invention there is providedplasmid pNG3-Vkss-HuCk-NEO deposited as NCIMB deposit no. 40799.

Plasmid pNG3-Vkss-HuCk-NEO was deposited at The National Collections ofIndustrial and Marine Bacteria (NCIMB), 23 St Machar Drive, Aberdeen AB21RY, Scotland, United Kingdom on Apr. 11, 1996 under deposit referencenumber NCIMB 40799 in accordance with the Budapest Treaty.

According to another aspect of the present invention there is provided amethod of making at least a variable region of a heavy or light chain ofan anti-CEA antibody as herein defined comprising:

a) transforming a host cell with a polynucleotide sequence which encodesat least the variable region of the heavy or light chain of the anti-CEAantibody and optionally developing the transformed host cell into atransgenic non-human mammal or transgenic plant;

b) subjecting the host cell, transgenic non-human mammal or transgenicplant to conditions conducive to expression, and optionally secretion,of at least the variable region and optionally;

c) at least partially purifying the variable region.

According to another aspect of the present invention there is provided amethod of making an antibody or a conjugate as defined herein whichcomprises:

a) subjecting a host cell, a transgenic non-human mammal or a transgenicplant as defined herein, or the 806.077 hybridoma, to conditionsconducive to expression, and optionally secretion, of the antibody orconjugate; and optionally

b) at least partially purifying the antibody or conjugate.

Preferably both heavy and light chain variable regions are expressed inthe same cell and assembled thereby to form an anti-CEA antibody.Preferably the heavy or light chain variable region is fused (optionallyvia some linking sequence) to a gene encoding a protein effector moiety(as part of a conjugate, see text below), preferably fusion is throughthe antibody heavy chain. Generally fusion can be either at the N or Cterminus of the antibody chain. For B7 conjugates fusion at theN-terminus of the antibody chain is preferred. CPB has an N-terminal prodomain which is believed to assist correct folding of protein before thepro domain is removed to release active enzyme. If proCPB is fused atits C terminus to the N terminus of an antibody chain this allowsremoval of pro domain (e.g. by trypsin treatment) from the N terminus ofthe fusion construct. Alternatively if proCPB was attached to the Cterminus of an antibody chain then the problem arises of having toremove the pro domain from the “middle” of the construct withoutdestroying the fusion protein. The solution is to co-express the prodomain separately (in trans). This has the advantage, once the celllines have been constructed, of not requiring trypsin activation ofexpressed fusion protein to remove CPB pro domain. Constructs withproCPB fused at its C terminus to the N terminus of an antibody chainhave the advantage of not requiring construction of co-expression celllines which require high level expression of the pro domain along withhigh level expression of other proteins.

According to another aspect of the present invention there is provided amethod of making monoclonal antibody 806.077 comprising:

a) culturing hybridoma 806.077 antibody deposited as ECACC deposit no.96022936 in medium under conditions conducive to expression of antibodytherefrom and;

b) obtaining antibody 806.077 antibody from the culture medium andoptionally;

c) preparing a F(ab′)₂ fragment of antibody 806.077 antibody by enzymicdigestion.

According to another aspect of the present invention there is provided aconjugate which comprises an effector moiety and an anti-CEA 806.077antibody of the invention as herein described. An effector moiety is anentity having the effect of bestowing another activity (e.g. an enzyme,toxin or radioactive ligand) to the 806.077 antibody in forming theconjugate.

In one embodiment, preferably the effector moiety is an enzyme suitablefor use in an ADEPT system. In International Patent Application WO96/20011, published Jul. 4, 1996, we proposed a “reversed polarity”ADEPT system based on mutant human enzymes having the advantage of lowimmunogenicity compared with for example bacterial enzymes. A particularhost enzyme was human pancreatic CPB (see for example, Example 15[D253K]human CPB & 16 [D253R]human CPB therein) and prodrugs therefor(see Examples 18 & 19 therein). The host enzyme is mutated to give achange in mode of interaction between enzyme and prodrug in terms ofrecognition of substrate compared with the native host enzyme. In oursubsequent International Patent Application No PCT/GB96/01975 (publishedMar. 6, 1997 as WO 97/07796) further work on mutant CPB enzyme/prodrugcombinations for ADEPT are described. Preferred enzymes suitable forADEPT are any one of CPG2 or a reversed polarity CPB enzyme, for exampleany one of [D253K]HCPB, [G251T,D253K]HCPB or [A248S,G251T,D253K]HCPB.

806.077 Antibody conjugates also have application in tumourimmunotherapy. Accordingly in another preferred embodiment the conjugateeffector moiety is a co-stimulatory molecule, preferably theco-stimulatory molecule is B7, more preferably human B7.1 or B7.2 andespecially human B7.1. Preferably the conjugate is in the form of afusion protein, preferably in which the fusion protein is formed throughlinking a C-terminus of the co-stimulatory molecule to an N-terminus806.077 antibody chain, preferably via the antibody chain heavy chain,preferably in which the 806.077 antibody lacks an Fc antibody region,more preferably a F(ab′)₂ antibody fragment, more preferably theantibody is humanised or human. An especially preferred conjugate isdescribed in Example 104 below.

The use of antibody to target a co-stimulatory molecule to tumour cellsis predicted to bestow the function of antigen presenting to the tumourcells such that T-cells receive specific TCR stimulation from the tumourcell itself and a co-stimulatory signal from the antibody targetedmolecule. The use of human or humanised antibodies is preferred for thetreatment of human tumours because murine antibodies may evoke an immunereaction when used in man which might result in a reduction ineffectivness on repeat therapy. The use of a fusion protein combining atumour antigen binding region linked to the extracellular portion of aco-stimulatory molecule is novel. Hayden et al (Tissue Antigens, 1996,48, 242-254) have reported the use of a bi-specific antibody moleculecombining an anti-tumour antigen binding domain with an anti-CD28binding domain. Whilst this molecule is capable of interacting with CD28on T-cells, the signal it may deliver has the disadvantage of beingqualitatively different from that provided by the natural CD28 ligands,for example the affinity of binding is greater than that between B7.1and CD28. The cross-species homology between B7.1,B7.2 and CD28,CTLA-4indicates evolutionary conservation of binding region sequences.Consequently it is believed that, for example B7.1 from man can interactwith CD28 from mouse and may impart a similar co-stimulatory signal. Fortreatment of human disease a human or humanised protein is preferable.However, the use of a human or humanised protein in animal models couldproduce similar effects to that anticipated in man and such animalmodels should provide relevant data as to the efficacy of ahuman/humanised antibody fusion protein with human B7.1/B7.2 in thetreatment of human disease.

Conjugation of the effector moiety and antibody may be by any suitablemethod such as for example chemical linkage via heterobifunctionallinkers or recombinant gene fusion techniques. In general fusionproteins are preferred conjugates, particularly for conjugates with HCPBor B7.

Preferred conjugates are those in which the effector moiety is selectedfrom any one of the following:

a) an enzyme suitable for use in an ADEPT system;

b) CPG2;

c) [G251T,D253K]HCPB;

d) [A248S,G251T,D253K]HCPB;

e) a co-stimulatory molecule;

f) extracellular domain of B7;

g) extracellular domain of human B7.1; and

h) extracellular domain of human B7.2;

optionally in the form of a fusion protein.

It will be appreciated that the conjugate of the present invention doesnot necessarily consist of one effector molecule and one antibodymolecule. For example the conjugate may comprise more than one effectormolecule per antibody molecule. In general, F(ab′)₂ antibody conjugateswhich are fusions between the antibody and an enzyme or an extracellulardomain of B7 will have 2 moles of enzyme or B7 per mole of antibody.

Especially preferred conjugates are a fusion protein selected from anyone of the following conjugates, (sequences being listed in N terminusto C terminus direction):

a) a humanised 806.077 F(ab′)₂-{[A248S,G251T,D253K]HCPB}₂ fusioncomprising:

an antibody Fd′ chain of structure VH1(SEQ ID NO: 55)/CH1 constantregion from IgG3/hinge region from IgG3;

the Fd′ chain being fused via its C terminus to the N terminus of[A248S,G251T,D253K]HCPB; and

an antibody light chain of formula VK4(SEQ ID NO: 71 )/CL region fromkappa light chain:

b) {[A248S,G251T,D253K]HCPB}₂-humanised 806.077 F(ab′)₂ fusioncomprising: [A248S,G251T,D253K]HCPB;

the HCPB being fused at its C terminus, via a (GGGS)₃ linker, to the Nterminus of an antibody Fd′ chain of structure VH1(SEQ ID NO: 55)/CH1constant region from IgG3/hinge region from IgG3; and

an antibody light chain of formula VK4(SEQ ID NO: 71)/CL region fromkappa light chain; and

c) a (human B7.1 extracellular domain)₂-humanised 806.077 F(ab′)₂ fusioncomprising: human B7.1 extracellular domain;

the B7.1 being fused at its C terminus to the N terminus of an antibodyFd′ chain of structure VH1(SEQ ID NO: 55)/CH1 constant region fromIgG3/hinge region from IgG3; and

an antibody light chain of structure VK4(SEQ ID NO: 71)/CL region fromkappa light chain.

In this specification the antibody hinge region in relation toconjugates is defined according to the principles set out by Padlan(1994) in Molecular Immunology 31. 169-217: see Table 2 therein inparticular. In these especially preferred conjugates there are 2 molesof enzyme or B7.1 per mole of F(ab′)₂. The forward slash (“/”) is merelya separator to indicate discrete structural elements joined by peptidebonds that make up parts of the conjugate.

The VH1 and/or VK4 variable region humanised sequences have theadvantage of preserving good binding properties with minimal additionalchanges required to the human framework. The IgG3 hinge region has theadvantage of giving good F(ab′)₂ production levels and homogeneity ofproduct.

In another preferred embodiment relating to the especially preferredconjugates defined above, fusion is effected through the antibody lightchain. In yet another preferred embodiment relating to the especiallypreferred conjugates defined above, the CH1 constant region fromIgG3/hinge region from IgG3 structural element may be replaced by thecorresponding IgG2 element.

When the effector molecule is a toxin, this toxin moiety generallycomprises a component which possesses cytotoxic properties and hence iscapable of killing cells following internalisation.

The toxin moiety and the anti-CEA antibody may be coupled directly toone another, or they may be coupled indirectly. The toxin moiety and theanti-CEA antibody are, in general, coupled such that the geometry of theconjugate permits the anti-CEA antibody to bind to its target cell.Advantageously, the toxin moiety and the anti-CEA antibody are coupledsuch that the conjugate is extracellularly stable, and intracellularlyunstable so that the toxin moiety and the anti-CEA antibody remaincoupled outside the target cell, but following internalisation, thetoxin moiety is released. Thus, advantageously the conjugate has anintracellularly cleavable/extracellularly stable site.

Examples of conjugates in which the toxin moiety is directly coupled tothe target cell binding moiety include those in which the toxin moietyand the anti-CEA antibody are coupled by a disulphide bridge formedbetween a thiol group on the toxin moiety and a thiol group on theanti-CEA antibody. Details of the preparation and properties ofimmunotoxins and other conjugates are given in European patentapplication EP 528 527 (publication no.) the contents of which isincorporated herein by reference thereto.

According to another aspect of the present invention there is provided apolynucleotide sequence capable of encoding a polypeptide of an antibodyor a conjugate as defined in any preceding claim. The term “capable ofencoding” is intended to encompass polynucleotide sequences taking intoaccount degeneracy in the genetic code in that some amino acids areencoded by more than one codon.

According to another aspect of the present invention there is provided avector comprising a polynucleotide as defined above.

According to another aspect of the present invention there is provided ahost cell transformed with a polynucleotide sequence as defined above ora transgenic non-human animal or transgenic plant developed from thehost cell.

According to another aspect of the present invention there is provided apharmaceutical composition comprising a conjugate of the inventiondescribed herein in association with a pharmaceutically-acceptablediluent or carrier, optionally in a form suitable for intravenousadministration.

According to another aspect of the present invention there is provided aconjugate as described herein for use as a medicament.

According to another aspect of the present invention there is provideduse of a conjugate as described herein for preparation of a medicamentfor treatment of neoplastic disease.

It will be appreciated that the dose and dosage regimen will depend uponthe particular effector moiety employed, the population of the targetcell and the patient's history. The dose of the conjugate administeredwill typically be in the range 0.1 to 1 mg/kg of patient weight.

The conjugates of the present invention will normally be administered inthe form of a pharmaceutical composition. Thus according to the presentinvention there is also provided a pharmaceutical compostion whichcomprises a conjugate (as defined herein) in association with apharmaceutically-acceptable diluent or carrier. An example of such aformulation is given herein in Example 10.

Pharmaceutical compositions of the present invention may be formulatedin a variety of dosage forms. Generally, the conjugates of the presentinvention will be administered parenterally, preferably intravenously. Aparticular parenteral pharmaceutical composition is one which isformulated in a unit dosage form which is suitable for administration byinjection. Thus, particularly suitable compositions comprise a solution,emulsion or suspension of the immunotoxin in association with apharmaceutically acceptable parenteral carrier or diluent. Suitablecarriers or diluents include aqueous vehicles, for example water orsaline, and non-aqueous vehicles, for example fixed oils or liposomes.The compositions may include agents which enhance the stability of theconjugate in the composition. For example, the composition may include abuffer. The concentration of the conjugate will vary, but in general,the conjugate will be formulated at concentrations of about 1 to 10mg/ml.

According to another aspect of the present invention there is providedan expression vector coding for an anti-CEA antibody of the invention asherein defined.

According to another aspect of the present invention there is providedan expression vector encoding at least the variable region of a heavy orlight chain of an anti-CEA antibody as herein defined.

According to another aspect of the present invention there is provided ahost cell transformed with a vector as herein described which iscompatible with expression therein.

According to another aspect of the present invention there is provided ahost cell transformed with a polynucleotide sequence as herein defined.

Mammalian cells (CHO, COS, myeloma) have been used as host for theco-expression of antibody H and L chain cDNAs and fragments thereof toproduce antibody with the specified binding activity (Bebbington, C.,1991, Methods, vol 2, p136-145, and Adair, J., 1992, ImmunologicalReviews, vol 130). For expression of constructs leading to directexpression of active CPB, COS or CHO cell expression systems arepreferred. The cDNAs can be introduced on plasmids and allowed tointegrate into chromosomal DNA especially for CHO cells or allowed toreplicate to very high copy number especially in COS cells. The plasmidsgenerally require a selectable marker for maintenance in transfectedhosts, an efficient eukaryotic promoter to allow a high level oftranscription from the cDNAs, convenient restriction enzyme sites forcloning and polyadenylation and transcription termination signals formessage stabilty. Several such vectors have been described in theliterature (Bebbington, C. et al, 1992, Bio/Technology, vol 10,p169-175, and Wright, A., 1991, Methods, vol 2, p125-135) and there arecommercially available vectors, (such as pRc/CMV ,Invitrogen Corp.)which are suitable.

The expression of a range of antibody fragments in E.coli is welldocumented (reviewed by Pluckthun, A., Immunological Reviews, 1992, vol130, p151-188 and Skerra, A., Current Opinion in Immunology, 1993, vol5, p256-262). Intracellular expression of Fd and L chains has beendescribed (Cabilly, S., 1989, Gene. vol 85, p553-557) but this mayrequire in vitro refolding and re-association of the chains (Buchner, Jand Rudolph, R., 1991, Bio/Technology, vol 9, p157-162) to producebinding activity. A more efficient route to obtaining active antibodyfragments is through periplasmic secretion (Better, M. et al, 1988,Science, vol 240, p1041-1043). The H and L chain components of theantibody fragment are co-expressed from a single plasmid. Each antibodychain is provided with a bacterial leader peptide which directs it tothe E.coli periplasm where the leader is cleaved and the free chainsassociate to produce soluble and active antibody fragments. This processis believed to mimic the natural process in eukaryotic cells where theexpressed antibody chains pass into the lumen of the endoplasmicreticulum prior to association into whole antibodies. This process oftenresults in the presence of binding activity in the culture supernatant.

Some expression systems involve transforming a host cell with a vector;such systems are well known such as for example in E. coli, yeast andmammalian hosts (see Methods in Enzymology 185, Academic Press 1990).Other systems of expression are also contemplated such as for exampletransgenic non-human mammals in which the gene of interest, preferablycut out from a vector and preferably in association with a mammarypromoter to direct expressed protein into the animal's milk, isintroduced into the pronucleus of a mammalian zygote (usually bymicroinjection into one of the two nuclei (usually the male nucleus) inthe pronucleus) and thereafter implanted into a foster mother. Aproportion of the animals produced by the foster mother will carry andexpress the introduced gene which has integrated into a chromosome.Usually the integrated gene is passed on to offspring by conventionalbreeding thus allowing ready expansion of stock. Preferably the proteinof interest is simply harvested from the milk of female transgenicanimals. The reader is directed to the following publications: Simons etal. (1988), Bio/Technology 6:179-183; Wright et al. (1991)Bio/Technology 9:830-834; U.S. Pat. No. 4,873,191 and; U.S. Pat. No.5,322,775. Manipulation of mouse embryos is described in Hogan et al,“Manipulating the Mouse Embryo; A Laboratory Manual”, Cold Spring HarborLaboratory 1986.

Transgenic plant technology is also contemplated such as for exampledescribed in the following publications: Swain W.F. (1991) TIBTECH 9:107-109; Ma J. K. C. et al (1994) Eur. J. Immunology 24: 131-138; HiattA. et al (1992) FEBS Letters 307:71-75; Hein M. B. et al (1991)Biotechnology Progress 7: 455-461; Duering K. (1990) Plant MolecularBiology 15: 281-294.

If desired, host genes can be inactivated or modified using standardprocedures as outlined briefly below and as described for example in“Gene Targeting; A Practical Approach”, IRL Press 1993. The target geneor portion of it is preferably cloned into a vector with a selectionmarker (such as Neo) inserted into the gene to disrupt its function. Thevector is linearised then transformed (usually by electroporation) intoembryonic stem (ES) cells (eg derived from a 129/Ola strain of mouse)and thereafter homologous recombination events take place in aproportion of the stem cells. The stem cells containing the genedisruption are expanded and injected into a blastocyst (such as forexample from a C57BL/6J mouse) and implanted into a foster mother fordevelopment. Chimaeric offspring can be identified by coat colourmarkers. Chimeras are bred to ascertain the contribution of the ES cellsto the germ line by mating to mice with genetic markers which allow adistinction to be made between ES derived and host blastocyst derivedgametes. Half of the ES cell derived gametes will carry the genemodification. Offspring are screened (eg by Southern blotting) toidentify those with a gene disruption (about 50% of progeny). Theseselected offspring will be heterozygous and therefore can be bred withanother heterozygote and homozygous offspring selected thereafter (about25% of progeny). Transgenic animals with a gene knockout can be crossedwith transgenic animals produced by known techniques such asmicroinjection of DNA into pronuclei, sphaeroplast fusion (Jakobovits etal. (1993) Nature 362:255-258) or lipid mediated transfection (Lamb etal. (1993) Nature Genetics 5 22-29) of ES cells to yield transgenicanimals with an endogenous gene knockout and foreign gene replacement.

ES cells containing a targeted gene disruption can be further modifiedby transforming with the target gene sequence containing a specificalteration, which is preferably cloned into a vector and linearisedprior to transformation. Following homologous recombination the alteredgene is introduced into the genome. These embryonic stem cells cansubsequently be used to create transgenics as described above.

The term “host cell” includes any procaryotic or eucaryotic cellsuitable for expression technology such as for example bacteria, yeasts,plant cells and non-human mammalian zygotes, oocytes, blastocysts,embryonic stem cells and any other suitable cells for transgenictechnology. If the context so permits the term “host cell” also includesa transgenic plant or non-human mammal developed from transformednon-human mammalian zygotes, oocytes. blastocysts, embryonic stem cells,plant cells and any other suitable cells for transgenic technology.

According to another aspect of the present invention there is provided amethod of treatment of a human or animal in need of such treatment whichcomprises administration to a human or animal of a pharmaceuticallyeffective amount of a conjugate as herein described.

According to another aspect of the present invention there is provided amethod of targeting an effector moiety to cells displaying antigen CEAin a mammal in need of such targeting which comprises administration ofa pharmaceutically effective amount of an conjugate of the invention asherein defined.

According to another aspect of the present invention there is providedthe use of an antibody as hereinbefore described in a diagnostic method.

One diagnostic method is immunoassay. An immunoassay for in vitrotesting based upon the novel antibody according to the invention may bedesigned in accordance with conventional immunological techniques in theart, utilising the antibody according to the invention in a labelled orunlabelled form and determining the complex formation of the antibodywith CEA in the sample to be tested. In one case, the antibody may belabelled with a detectable label, such as radiolabel, a chemiluminescer,a fluorescer or an enzyme label. Alternatively the antibody is detectedvia a complex formed with a labelled substance or by non-labellingtechniques, such as biosensor methods eg based upon surface plasmonresonance. The sample may, for example, be in the form of a body fluid,such as serum, or a tissue preparation (histochemical assay).

For in vivo diagnostic purposes, the antibody according to the inventionis provided with a suitable externally detectable label, such as eg. aradiolabel or a heavy metal atom, and administered to a subjectwhereupon the possible localised accumulation of antibody in the body isdetermined.

For the in vitro diagnosis of cancer the anti-CEA antibody can beconjugated to either enzymes such as horse radish peroxidase andbacterial luciferase which can generate a signal which can be measuredor to fluorescent markers or radioisotopes which can be detected andquantitated directly. In a standard immunoassay system such conjugatesprovide a means of measuring the presence or absence of CEA in bodytissues and consequently provides a rapid and convenient test for thediagnosis of tumour disease. See general descriptions of the methodologyinvolved in Enzyme immunoassay, E. T. Maggio, CRC Press and U.S. Pat.No. 3690 8334, U.S. 3,791,932, U.S. 3,817,837, U.S. 3,850,578, U.S.3,853,987, U.S. 3,867,517, U.S. 3,901,654, U.S. 3,935,074, U.S.3,984,533, U.S. 3,996,345 and U.S. 4,098,876.

For the in vivo diagnosis of cancer, the anti-CEA antibody can beconjugated to isotopes of elements such as yttrium, technetium or indiumor heavy metal isotopes which can be detected by whole body imagingcameras (see Larson, S. M. ,1987, Radiology, 165, 297-304.

For the therapy of cancer, preferred embodiments involve an anti-CEAantibody that can be conjugated to an effector moiety which can kill thecancer cells directly or especially via activation of a suitable prodrugin an ADEPT system. In ADEPT selective killing of tumour cells isachieved by conjugating the anti-CEA antibody to an enzyme which iscapable of catalysing the conversion of a non-toxic dose of a prodruginto a potent toxic drug compound. Administration of the conjugate leadsto localization of the enzyme activity at the tumour site. Subsequentadministration of the prodrug leads to local production of the toxicdrug and selective kill at the tumour site. This approach is describedin WO 88/07378, U.S. Pat. No. 4,975,278, U.S. 5,405,990 and WO89/10140.Antibody 806.077 may also be used conjugated to a co-stimulatorymolecule for tumour immunotherapy as described above.

Selective cell killing of tumour cells can also be achieved byconjugation of the anti-CEA antibody either directly or by chemicalderivatization with macrocycle chelators containing high energyradioisotopes such as ⁹⁰Y, ¹³¹I and ¹¹¹In. The anti-CEA antibody servesto localize the isotope to the tumour and the radiation emitted by theisotope destroys the DNA of the surrounding cells and kills the tumour.

Selective killing of tumour cells can also be achieved by conjugation ofthe anti-CEA antibody to cytotoxic and cytostatic drugs such asmethotrexate, chlorambucil, adriamycin, daunorubicin and vincristine.These drugs have been used in the clinic for many years and the therapythey provide is often limited by non specific toxicity. Conjugation ofthese drugs to the CEA antibody enables these drugs to localize at thetumour site and thus increasing the dose of drug that can be deliveredto the tumour without incurring unacceptable side effects from theaction of such drugs on other tissues such as the bone marrow or nervoussystem. The effectiveness of the antibody is in many applicationsimproved by reducing the size of the antibody binding structure andthereby improving the tissue penetration and other pharmacodynamicproperties of the pharmaceutical composition. This can be achieved byremoving the Fc region of the antibody molecule either enzymically or bygenetic engineering methods to produce a recombinant Fab′ or F(ab′)₂fragment.

Genetic engineering methods can also be used to further reduce the sizeof the anti-CEA antibody. The Fv which contain the CDRs can beengineered and expressed in isolation and chemically cross linked forinstance by the use of disulphide bridges. Alternatively, both the lightand heavy chain domains making up the Fv structure may be produced as asingle polypeptide chain (SCFv) by fusing the Fv domains with a linkerpeptide sequence from the natural C-terminus of one domain to theN-terminus of the other domain (see PCT/US/87/02208 and U.S. Pat. No.4,704,692). Alternatively, a single Fv domain may be expressed inisolation forming a single domain antibody or dAb as described by Wardet al Nature(1989) 341, 544. Another type of anti-CEA antibodycontemplated is a V-min construct as disclosed in International PatentApplication WO 94/12625 (inventors Slater & Timms). Abbreviations usedherein include:

ADEPT antibody directed enzyme prodrug therapy APC antigen presentingcell CDRs complementarity determining regions CEA Carcinoma EmbryonicAntigen CL constant domain of antibody light chain CPB carboxypeptidaseB CPG2 carboxypeptidase G2 DAB substrate 3,3′-diaminobenzidinetetrahydrochloride DEPC diethylpyrocarbonate DMEM Dulbecco's modifiedEagle's medium ECACC European Collection of Animal Cell Cultures EIAenzyme immunoassay ELISA enzyme linked immunosorbent assay FCS foetalcalf serum Fd heavy chain of Fab, Fab′ or F(ab′)₂ optionally containinga hinge HAMA Human Anti Mouse Antibody HCPB human carboxypeptidase B,preferably pancreatic hinge (of an IgG) a short proline rich peptidewhich contains the cysteines that bridge the 2 heavy chains HRPO horseradish peroxidase NCA non-specific cross reacting antigen NCIMB NationalCollections of Industrial and Marine Bacteria PBS phosphate bufferedsaline PCR polymerase chain reaction preproCPB proCPB with an N-terminalleader sequence proCPB CPB with its N-terminal pro domain SDS-PAGEsodium dodecyl sulphate - polyacrylamide gel electrophoresis TBSTris-buffered Saline VH variable region of the heavy antibody chain VKvariable region of the light antibody chain

The invention is illustrated by the following non-limiting Examples(supported by Reference Examples which follow the Examples) in which:

FIG. 1 shows anti-tumour activity of 806.077 antibody-CPG2 conjugate inan ADEPT model;

FIG. 2 shows a plasmid map of pCF009;

FIG. 3 shows BIAcore data showing antibody-B7.1 fusion protein bindingto immobilised CTLA4-Ig in which the solid line represents test bindingand the dotted line is a blank control; and unless otherwise stated;

DNA is recovered and purified by use of GENECLEAN™ II kit (StratechScientific Ltd. or Bio 101 Inc.). The kit contains: 1) 6M sodium iodide;2) a concentrated solution of sodium chloride, Tris and EDTA for makinga sodium chloride/ethanol/water wash; 3) Glassmilk a 1.5 ml vialcontaining 1.25 ml of a suspension of a specially formulated silicamatrix in water. This is a technique for DNA purification based on themethod of Vogelstein and Gillespie published in Proceedings of theNational Academy of Sciences USA (1979) Vol 76, p 615. Briefly, the kitprocedure is as follows. To 1 volume of gel slice is added 3 volumes ofsodium iodide solution from the kit. The agarose is melted by heatingthe mix at 55° C. for 10 min then Glassmilk (5-10 ml) is added, mixedwell and left to stand for 10 min at ambient temperature. The glassmilkis spun down and washed 3 times with NEW WASH (0.5 ml) from the kit. Thewash buffer is removed from the Glassmilk which is to dry in air. TheDNA is eluted by incubating the dried Glassmilk with water (5-10 ml) at55° C. for 5-10 min. The aqueous supernatant containing the eluted DNAis recovered by centrifugation. The elution step can be repeated andsupernatants pooled;

Competent E. coli DH5α cells were obtained from Life Technologies Ltd(MAX efficiency DH5α competent cells);

Mini-preparations of double stranded plasmid DNA were made using theRPM™ DNA preparation kit from Bio101 Inc. (cat. No 2070-400) or asimilar product—the kit contains alkaline lysis solution to liberateplasmid DNA from bacterial cells and glassmilk in a spinfilter to adsorbliberated DNA which is then eluted with sterile water or 10 mM Tris-HCl,1 mM EDTA, pH 7.5;

Serum free medium is OPTIMEM™ I Reduced Serum Medium, GibcoBRL Cat. No.31985;

LIPOFECTIN™ Reagent (GibcoBRL Cat. No. 18292-011) is a 1:1 (w/w)liposome formulation of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA)and dioleoyl phosphatidylethanolamine (DOPE) in membrane filtered water.It binds spontaneously with DNA to form a lipid-DNA complex—see Felgneret al. in Proc. Natl. Acad. Sci. USA (1987) 84, 7431;

G418 (sulphate) is GENETICIN™, GibcoBRL Cat. No 11811, an aminoglycosideantibiotic related to gentamicin used as a selecting agent in moleculargenetic experiments;

AMPLITAQ™ available from Perkin-Elmer Cetus, is used as the source ofthermostable DNA polymerase; and

General molecular biology procedures can be followed from any of themethods described in “Molecular Cloning—A Laboratory Manual” SecondEdition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory,1989).

EXAMPLE 1

Discovery and Establishment of Hybridoma Cell Line 806.077

BALB/C mice, 8 to 10 weeks old, were immunised subcutaneously with aprimary dose of CEA (10 μg) in phosphate buffered saline solution (0.1ml) and Freund's Complete adjuvant (0.1 ml). Two weeks later and again 2weeks later the animals were boosted with further doses of CEA (10 μg)in phosphate buffered saline (0.1 ml) mixed with Freund's Incompleteadjuvant (0.1 ml). Thirty two weeks later the animals were given a finalintravenous immunisation of CEA (10 μg) in phosphate buffered saline andsacrificed three days later. The spleens were removed and prepared andfused with NS0 cells (available from the European Collection of AnimalCell Cultures under the accession No. 85110503) by standard methods(Kohler and Milstein, Nature (1975) 256, 495). The resulting cells weredistributed into 96-well culture dishes and incubated for 2 weeks. Thesupernatants from the resulting hybridomas were screened by EIA (enzymeimmunoassay). From a total of 1,824 wells generated from 5 fusions, 102wells were positive against native CEA. In fusion 806, seventeen wellswere found to be positive. The cells contained in these wells werecloned by limiting dilution, and the resulting clones tested by EIA.Lines from 10/17 original wells cloned successfully. One line,designated 806.077, has been deposited with the European Collection ofAnimal Cell Cultures under Accession No. 96022936. The table belowprovides a summary of the antibody generation programme that led todiscovery of the 806.077 antibody hybridoma.

number number CEA* number rest number Wells +ve by finally Antigenhousing weeks fusions tested EIA selected Untreated normal 8-20 2813,920  99 0 CEA desialated normal 8-12 14 5,568  12* 0 CEA conjugatednormal 10-12   8 3,168  1 0 CEA Untreated isolator >30  5 1,824 102  3CEA *tested against untreated CEA desialated immunisations produced lotsof +ves when tested against the immunogen

EXAMPLE 2

Preparation of 806.077 Antibody from Deposited Hybridoma Cell Line ECACCNo. 96022936

2.1 Preparation from Serum Containing Medium

A 1 ml cryopreserved ampoule was removed from storage in liquid nitrogenand rapidly thawed in a 37° C. water bath. The contents were asepticallytransferred to a sterile 15 ml centrifuge tube. The cells wereresuspended by dropwise addition of 10 ml of Dulbecco's modified Eagle'smedium (DMEM) containing 10% (v/v) foetal calf serum (FCS) accompaniedby gentle mixing. The suspension was centrifuged at 50×g for 10 min, thesupernatant aseptically removed and the pellet resuspended in 5 ml ofDMEM, 10% FCS and 1% L-glutamine in a 95% air 5% carbon dioxidepre-gassed 25 ml tissue culture flask. The flask was incubated at 36.5°C. in the dark.

After 3 days the flask was sub-cultured by passing the contents of theentire flask into a larger 75 ml flask diluting with DMEM, 10% FCS and1% L-glutamine (final viable density=2-3×10⁵ cells/ml). Furtherexpansion to 162 ml flasks was performed in a similar manner.

Culture supernatants for purification were prepared in 500 ml rollercultures in 850 ml roller bottles. Cultures were seeded at 2×10⁵ viablecells/ml in pre-gassed roller bottles, rotated at 3 rpm and incubated at36.5° C. Cultures were grown to maturity and harvested typically 500-800hours after inoculation when the cell viability was below 10% and IgGconcentration had reached a maximum.

2.2 Treatment of Culture Harvests

After harvest, roller bottle culture supernatants were clarified bycentrifugation at 60×g for 30 minutes. Sodium azide (0.02% w/v) wasadded as a preservative to the clarified supernatant which was stored at4° C. in the dark until purification.

2.3 Purification of 806.077 Antibody

806.077 Antibody hybridoma supernatant (3 l) was adjusted to pH 7.5 withdilute aqueous sodium hydroxide and filtered through a 0.45 cm filter(Millipore MILLIDISK™). The filtered antibody supernatant was loadedonto an affinity column of Protein G (for example Protein G Fast FlowSEPHAROSE™, Pharmacia product code 17.0618.03; 5 cm i.d×6.5 cm=130 ml;)equilibrated in phosphate buffered saline (“PBS”; 8 mM Na₂HPO₄, 1.5 mMKH₂PO₄, 150 mM NaCl, 2.5 mM KCl, pH 7.3, for example as available intablet form for reconstitution from Oxoid) at 4° C. at a flow-rate of 4ml/min. The column was washed with PBS (260 ml) at the same flow rateand the antibody eluted with 100 mM sodium citrate pH 2.6, collectingfractions and monitoring the eluate by UV absorption (280 nm). The UVabsorbing fractions containing the antibody, were bulked, immediatelyadjusted to pH 7 and concentrated to about 2 mg/ml by ultrafiltrationusing a 30 kDa cut-off membrane (e.g. Amicon YM30). Dialysis, using a6-8 kDa porosity cut-off membrane (e.g. SPECTRAPOR™ 1) membrane, into 50mM tris-HCl pH 7.0 buffer yielded 110 mg 806.077 antibody, >95% pure bySDS-PAGE.

EXAMPLE 3

Selectivity of 806.077 Antibody

To assess selectivity, many human normal and tumour tissues have beenscreened for reactivity with the antibody 806.077, using sensitivethree-stage indirect immunohistology on acetone-fixed, frozen cryostatsections.

Immunohistology was carried out on sections of human tissue obtainedeither at resection surgery or at post mortem. To preserve optimalmorphology and antigenicity, tissues were obtained as fresh as possible,cut into small pieces (about 0.5 cm³) and flash frozen in liquidnitrogen prior to storage at −80° C. Sections of tissue (6μ) were cut ona cryostat, mounted on polylysine coated slides (e.g. blue TECHMATE™slides, Dako) and fixed in ice-cold acetone for 2 minutes before beingwrapped in foil and stored at −80° C.

Slides were allowed to defrost at room temperature before beingunwrapped from the foil immediately prior to use. Each section wasoutlined with a diamond marker, and to each section was added either 100μl 806.077 antibody diluted to 2 μg/ml in Tris-buffered Saline (TBS), or100 μl CEA/NCA reactive control (A5B7 antibody) at 2 μg/ml in TBS, or100 μl MOPC isotype control (Sigma Chemical Company, St. Louis, U.S.A.,Cat. No. M 9269) at 2 μg/ml in TBS, or relevant positive control such asLP34 (Dako). All subsequent incubations were carried out at roomtemperature for 30 minutes in a humidified chamber: all wash steps werein TBS with 2 changes. After incubation, the slides were washed and 100μl of second antibody reagent, comprising {fraction (1/50)} rabbitanti-mouse immunoglobulins conjugated to horse radish peroxidase (DakoPatts) and ⅕ normal human serum (Sigma) in TBS was added to eachsection.

The slides were again incubated and washed in TBS. A final detectingantibody, 100 μl swine anti-rabbit immunoglobulin conjugated to horseradish peroxidase (1150 dilution with 115 normal human serum in TBS),was added to each section, incubated and washed thoroughly. DABsubstrate (3,3′-diaminobenzidine tetrahydrochloride) was prepared using1 DAB tablet (Sigma) with hydrogen peroxide (17 μl) in TBS (17 ml), andadded dropwise through a fast filter paper (e.g. Whatman Number 4).After 3 minutes incubation the excess DAB was tapped off the slides andthe slides were washed in TBS. After counter staining with haematoxylin(e.g. Mayer's Haematoxylin, Shandon) sections were dehydrated in alcoholand xylene, and mounted in non-aqueous synthetic mountant (e.g. E-Zmountant, Shandon) before examination under a microscope.

The areas of antibody bonding were visualised by brown staining on thesection. A scoring system was used to evaluate the degree of binding of806.077 antibody to tissues:

+++ (strong) = antibody binding to >75% tumour cells ++ (moderate) =antibody binding to 50%-75% tumour cells + (weak) = antibody binding to25%-50% tumour cells +/− (minimal) = non-focal antibody binding to asmall area of tumour cells − = no staining

Carcinoembryonic antigen (CEA) is a member of the immunoglobulin genesuperfamily with one predicted variable-like domain region (N domain;108 amino acids) and three sets of constant domain-like regions A1B1,A2B2 and A3B3; 92 amino acids for A domains and 86 amino acids for Bdomains (Hefta, 1992, Cancer Research 52:5647-5655. In addition, CEApossesses two signal peptides, one at the amino terminus and one at thecarboxyl terminus. Both are removed during post-translationalprocessing, the one at the carboxy terminus being replaced by aglycosylphosphatidylinositol (GPI) moiety. A large number of CEA-relatedproteins with varying homology to CEA have been reported (Thompson,1991, J. of Clinical Laboratory Analysis, 5: 344-366). These includenon-specific cross reacting antigens, NCA 1 and 2. These relatedproteins are expressed on a range of normal tissues includinggranulocytes and normal lung epithelium. The majority of anti-CEAmonoclonal antibodies generated so far, cross react with one of theserelated proteins and thus react with a range of normal tissues and oftenreact strongly with either granulocytes or lung epithelium.

Anti-CEA antibody, 806.077 was identified as being CEA selective,exhibiting no cross reactivity to granulocytes and only minimal stainingto {fraction (4/14)} normal lung tissues tested. 806.077 antibody wasinitially screened for tumour and NCA selectivity as a tissue culturesupernatant. The screens were carried out using the supernatant neat anddiluted at 1:10 and demonstrated equivalent binding of the antibody tocolon tumours when compared to A5B7, but much reduced binding to normallung and spleen tissues when compared to the same antibody. The antibodywas affinity purified (as described in Example 2) and the screensrepeated and extended to include further tumours and tissue types.

The antibody was titrated against a panel of colo-rectal tumour sectionsand this screen demonstrated the optimum screening concentration of806.077 antibody to be 2 μg/ml. All subsequent screens were carried outusing the antibody at this concentration. The results of these screenswere as follows. The reactivity of 806.077 antibody was compared againstA5B7 (also screened at 2 μg/ml) against the following tumours/normaltissues:

806.077 Antibody Tumour Reactivity:

Colon tumours (n=17).

Moderate to strong reactivity (++/+++equivalent to A5B7) was seen to all17 tumours tested.

Breast tumours (n=6).

Moderate/weak staining (+/++), {fraction (2/6)} tumours; minimalstaining(+/−), {fraction (2/6)} tumours.

NSCLC tumours (n=6).

Strong staining (+++), {fraction (2/6)} tumours; moderate staining (++),⅙ and weak staining (+), {fraction (2/6)} tumours.

Gastric tumours (n=2).

Strong staining (+++), ½ tumours; weak staining (+) ½ tumours.

Ovary tumours (n=3) and prostate tumours (n=3).

No staining was seen to any of these tumours.

In all cases, equivalent reactivity was seen with A5B7.

Normal tissue reactivity:

Lung (NCA reactivity) (n=14).

Weak staining (+), {fraction (4/14)} lung tissues; no staining (−){fraction (10/14)} tissues.

A5B7 bound moderately (++), {fraction (1/14)} lung tissues; weakly (+),{fraction (10/14)} tissues and minimally (+/−), {fraction (1/14)}tissues.

Spleen (granulocyte/NCA reactivity) (n=6).

No staining was seen to any of the spleen tissues tested.

A5B7 bound moderately (++), ⅙ tissues and weakly (+), ⅚ tissues

Post mortem normal tissues (n=13).

Moderate/weak reactivity (++/+) was seen only to oesophagus, skin, colonand pancreas tissues (CEA expressing normal tissues). Similar bindingwas seen with A5B7. in addition to the positive tissues, colon, skin,oesophagus and pancreas, the negative tissues were: cerebellum,mid-brain, cerebrum, smooth muscle, liver, kidney, aorta, stomach,heart.

EXAMPLE 4

Generation of 806.077 Antibody F(ab′) Fragment

Ficin (10 mg) was suspended in a solution of 50 mM cysteine (3 ml; BDH37218) and 50 mM tris-HCl pH 7.0 and incubated at 37° C. for 30 minutes.Excess cysteine was removed by size exclusion chromatography (Sephadex™G-25 column, 1.5 cm×25 cm; Pharmacia) in 50 mM tris-HCl pH 7.0 buffer.The reduced ficin concentration was determined by monitoring UVabsorbance at A280 nm (assuming that a 1 mg/ml solution has anabsorbance reading of 2 in a 1 cm cell) and was found to be 1.65 mg/ml.

A solution of 806.077 antibody (100 mg) in 50 mM tris-HCl buffer pH 7.0(50 ml) and freshly reduced ficin (5 mg; 3 ml of the above solution) wasdigested at 37° C. over 20 hours. The digest was then diluted with anequal volume of PBS and loaded onto a Protein G affinity column(Pharmacia SEPHAROSE™ Fast Flow, 5.0 cm i.d×6.5 cm=125 ml; previouslyequilibrated with 50 mM tris-HCl pH 7.0 buffer at 4° C.), at a constantflow-rate of 3 ml/min. The column was washed with 50 mM sodium acetatepH 4.0 (250 ml) to remove low M.W. fragments, followed by 50 mM sodiumcitrate pH 2.8 to elute the F(ab′)₂, monitoring the UV adsorbance of theeluate at A280 nm. The F(ab′)₂ containing eluate was adjusted to pH 7and buffer exchanged into 100 mM sodium phosphate/100 mM sodiumchloride/1 mM EDTA pH 7.2 by dialysis and concentrated to 8 mg/ml bymembrane filtration using a 10 kDa cut-off (e.g. Amicon™ YM10) assumingthat a 1 mg/ml solution has an absorbance reading at 280 nm of 1.4 in a1 cm cell. A 65% yield of 42 mg 95% pure F(ab′)₂ was obtained.

EXAMPLE 5

Preparation of 806.077 Antibody F(ab′)₂—Carboxypeptidase G2 Conjugate

The linker for 806.077 antibody F(ab′)₂ derivatisation was SATA(S-acetyl thioglycollic acid N-hydroxysuccinimide ester, Sigma, productcode A 9043) The linker for carboxypeptidase G2 (CPG2) derivatisationwas SMPB [4-p-maleimidophenyl) butyric acid N-hydroxysuccinimide ester,Sigma, product code M6139]

5.1 F(ab′), Derivatisation:

To a solution of the F(ab′)₂ fragment (40 mg, prepared as described inExample 4) in 100 mM phosphate/100 mM NaCl/1 mM EDTA pH 7.2 (buffer A; 5ml) was mixed with SATA (0.28 mg) in DMSO (28 μl). After 40 minutes atroom temperature the resulting solution was applied to a desaltingcolumn (SEPHADEX™ G-25, 1.5 cm i.d×50 cm=100 ml; equilibrated in bufferA at 4° C.) at a flow-rate of 1.2 ml/min. to remove excess reagents. Theeluate was monitored by UV absorption at A280 nm. The SATA derivatisedF(ab′)₂ was pooled and mixed with 10% v/v 500 mM hydroxylamine HCl/500mM sodium phosphate/30 mM EDTA pH 8.0 for 60 minutes at room temperatureto deacetylate the derivatised F(ab′)₂. The protein concentration wasdetermined by UV absorption at 280 nm assuming that a 1 mg/ml solutionhas an absorbance reading of 1.4 in a 1 cm cell. The solution wasdiluted to about 1 mg/ml with buffer A. The linker loading wasdetermined by Eliman's -SH assay and found to be 1.8-2.0 linkers/moleF(ab′)₂.

5.2 CPG2 Derivatisation:

Large scale purification of CPG2 from Pseudomonas RS-16 was described inSherwood et al. (1985), Eur, J. Biochem., 148, 447-453. Preparation ofF(ab′)₂ and IgG antibodies coupled to CPG enzyme may be effected byknown means and has been described for example in PCT WO 89/10140. CPGmay be obtained from Centre for Applied Microbiology and Research,Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom. CPG2 may alsobe obtained by recombinant techniques. The nucleotide coding sequencefor CPG2 has been published by Minton, N. P. et al., Gene, (1984) 31,31-38. Expression of the coding sequence has been reported in E.coli(Chambers. S. P. et al., Appl. Microbiol, Biotechnol. (1988), 29,572-578) and in Saccharomyces cerevisiae (Clarke, L. E. et al., J. GenMicrobiol, (1985) 131, 897-904). Total gene synthesis has been describedby M. Edwards in Am. Biotech. Lab (1987), 5, 3844. Expression ofheterologous proteins in E.coli has been reviewed by F. A. O. Marston inDNA Cloning Vol. III, Practical Approach Series, IRL Press (Editor D MGlover), 1987, 59-88. Expression of proteins in yeast has been reviewedin Methods in Enzymology Volume 194, Academic Press 1991, Edited by C.Guthrie and G R Fink.

CPG enzyme is available from Sigma Chemical Company, Fancy Road, Poole,Dorset, U.K. CPG enzyme was described in: Goldman. P. and Levy, C. C.,PNAS USA, 58: 1299-1306 (1967) and in: Levy, C. C. and Goldman P., J.Biol. Chem., 242: 2933-2938 (1967). Carboxypeptidase G3 enzyme has beendescribed in Yasuda, N. et al., Biosci. Biotech. Biochem., 56: 1536-1540(1992). Carboxypeptidase G2 enzyme has been described in European Patent121 352.

CPG2 (50 mg; recombinant enzyme from E. coli) was dialysed into 100 mMsodium phosphate/100 mM sodium chloride pH 7.2 (=buffer B) and dilutedto 8 mg/ml, assuming that a 1 mg/ml solution has an absorbance readingat 280 nm of 0.6 in a 1 cm cell.

SMPB(Sigma) was dissolved in DMSO at 10 mg/ml. CPG2 (50 mg in buffer Bat 8 mg/ml) was mixed with the SMPB solution (0.108 ml; 1.08 mg), andreacted at room temperature for 120 minutes. Excess reagents wereremoved on a desalting column (Sephadex G-25, 1.5 cm i.d×50 cm=100 ml;equilibrated in buffer B at 4° C.) at 1.2 ml/min. Derivatised CPG2 waspooled and the concentration determined by V A280 nm, assuming that a 1mg/ml solution has an absorbance reading at 280 nm of 0.6 in a 1 cmcell. The solution was diluted to a CPG2 concentration of about 1 mg/ml.The linker loading was determined by a ‘reverse’ Ellman's assay, byadding a known amount of 2-mercaptoethanol to the maleimido-derivatisedCPG2 and assaying unreacted —SH. A linker loading of 2.0-2.4linkers/mole CPG2 was found.

5.3 Conjugation:

Equal weights of the deacetylated derivatised F(ab′)₂ and derivatisedCPG2 were mixed under nitrogen and the mixture (about 80 ml, at a totalprotein concentration of about 1 mg/ml) left at room temperature for 20h. The reaction was terminated by the addition of 10% v/v 100 mM aqueousglycine. The crude conjugation mixture was buffer exchanged by dialysisinto a low salt buffer (50 mM sodium acetate pH 6.0) and applied to adye-ligand affinity column (where the dye binds to CPG2 e.g. ACL MimeticGreen 1, 2.5 cm i.d×10 cm=50 ml) at 4° C. equilibrated in the samebuffer, to remove unreacted derivatised F(ab′)₂. The conjugate andderivatised CPG2 were eluted with 50 mM acetate/500 mM NaCl pH 6.0, at aflow rate of 2.0 ml/min monitoring the elution by UV (A280 nm).

The crude conjugate, still containing derivatised CPG2, was concentratedusing a 10 kDa cut-off ultrafiltration device (e.g. Amicon YM10™) toabout 12 ml, at 5 mg/ml total protein concentration and 10% v/v 10 mMzinc sulphate (Sigma Z 0251) in water was added to replenish zinc lostto the CPG2 in the process. Further chromatography by size exclusion(e.g. SEPHACRYL S-300HR™ Pharmacia, 2.5 cm i.d×25 cm=500 ml) at 4° C. in50 mM sodium acetate/150 mM sodium chloride pH 6.0 at a flow-rate of 1ml/min., collecting fractions and monitoring by UV A280 nm, resulted inthe fractionation of the conjugate and its separation from unreactedderivatised CPG2, as determined by SDS-PAGE of column fractions.

The peak containing conjugate (with ratios of F(ab′)₂:CPG2 of 1:2, 1:1and 2:1) was pooled and concentrated by ultrafiltration to 1.3 mg/ml,the protein concentration being determined by monitoring UV adsorbanceat A280 nm (assuming 1 mg/ml has an absorbance of 1.0). Purity of theconjugate was determined by SDS PAGE and found to contain a total of 12mg conjugate with the composition 65% 1:1 ratio conjugate, 20% 1:2 or2:1 ratio conjugate with <5% free derivatised F(ab′)₂ and <5% freederivatised CPG2.

EXAMPLE 6

Anti-Tumour Activity of 806.077 Antibody F(ab′)₂-CPG2 Conjugate inCombination with a Prodrug.

The anti-tumour activity of the 806.077 antibody F(ab′)₂-CPG2 conjugateprepared as described in Example 5 was evaluated in combination with theprodrug N(4-[N,N-bis(2-chloroethyl)amino]-phenoxycarbonyl)-L-glutamicacid (called “PGP” in this example, is described in Example 1 in U.S.Pat. No. 5,405,990 and Blakey et al., Br. J. Cancer 72, 1083-88, 1995)in a human colorectal tumour xenograft model.

Groups of 8-10 female athymic nude mice were injected s.c. with 1×10⁷LoVo colorectal tumour cells (ECACC no 87060101). When the tumours were4-5 mm in diameter either 806.077 antibody F(ab′)₂-CPG2 conjugate (250 UCPG2 enzyme activity Kg⁻¹) or phosphate buffered saline (170 mM NaCl,3.4 mM KCl, 12 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.2) was injectedintravenously (i.v). Seventy-two hours later PGP prodrug was injectedi.p. (3 doses of 40 mg/Kg at 1 h intervals). The length of the tumoursin two directions was then measured three times a week and the tumourvolume calculated using the formula:

Volume=π/6×D ² d

where D is the larger diameter and d is the smaller diameter of thetumour. Tumour volume was expressed relative to the tumour volume at thetime of initiation of the prodrug arm of the therapy. The anti-tumouractivity was compared with control groups given PBS instead of eitherconjugate or prodrug. Anti-tumour activity was expressed both as agrowth delay defined as the time it takes treated tumours to increasetheir volume by 4-fold minus the time it takes control tumours toincrease their volume 4-fold and as a T/C value defined as the volume ofthe treated tumour divided by the volume of the control tumour 14 daysafter prodrug administration. Statistical significance of theanti-tumour effects was judged using the analysis of variance (one-way)test.

The anti-tumour activity of 806.077 antibody F(ab′)₂-CPG2 conjugate incombination with PGP prodrug are shown in FIG. 1 and the anti-tumourdata is summarised below. Anti-tumour activity of 806.077 antibodyF(ab′)2-CPG2 conjugate in combination with PGP prodrug in LoVo tumourxenografts.

Growth Dose T/C delay Significance Conjugate (U/kg) (%) (days) (p)806.077 F(ab′)₂-CPG2 250 16.5 14 <0.01 500  4.7 22 <0.01

The results demonstrate that the 806.077 antibody F(ab′)₂-CPG2 conjugatein combination with the PGP prodrug produce tumour regressions andprolonged growth delays which were statistically significant comparedwith control groups.

EXAMPLE 7

Cloning and Sequencing of the Variable Regions of 806.077 Antibody Heavyand Light Chain Genes

7.1 Preparation of Cytoplasmic RNA

There are several procedures for the isolation of polyA+mRNA fromeukaryotic cells (Sambrook J., Fritsch E. F., Maniatis T., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Second Edition, 1989, Chapter 8, p3 herein referred to as “Maniatis”).In this particular case cytoplasmic RNA was prepared as described byFavoloro et al., Methods in Enzymology 65, 718-749, from a frozenhybridoma cell pellet containing 1×10⁹ cells which had been stored at−80° C.

The cells were resuspended in 5 ml ice-cold lysis buffer (140 mM NaCl,1.5 mM MgCl₂, 10 mM Tris-HCl pH 8.6 and 0.5% NP40 (a polyglycol ethernonionic detergent; Nonylphenoxy Polyethoxy Ethanol, Sigma Cat. No.127087-87-0)) containing 400 u of a ribonuclease inhibitor (RNAguard;Pharmacia Cat. No. 27-0815-01) and vortexed for 10 s. This solution wasoverlayed on an equal volume of ice cold lysis buffer containing 24%(w/v) sucrose and 1% NP-40 and stored on ice for 5 min. The preparationwas then centrifuged at 4000 rpm for 30 min at 4° C. in a bench topcentrifuge (Sorval RT6000B ) after which, the upper cytoplasmic phasewas removed to an equal volume of 2×PK buffer (200 mM Tris (pH7.5), 25mM EDTA, 300 mM NaCl and 2% (w/v) SDS). Proteinase K (Sigma, Cat No.P2308) was added to a final concentration of 200 μg/ml and the mixtureincubated at 37° C. for 30 min.

The preparation was extracted with an equal volume of phenol/chloroform,the aqueous phase removed and 2.5 vol ethanol added and mixed. Thissolution was then stored at −20° C. overnight. RNA was collected bycentrifugation (4000 rpm, 30 min at 4° C. in a bench top centrifuge,Sorval RT6000B), the supernatant decanted and the pellet dried in avacuum dessicator after which it was dissolved in 250 μldiethylpyrocarbonate (DEPC)-treated water (prepared as described inManiatis, referenced above). The RNA content was measured byspectrophotometry and the concentration calculated assuming anabsorbance at 260 nm of 1=40 μg/ml.

7.2 Preparation of First Strand Variable Region cDNA

A number of methods for the synthesis of cDNA are reviewed in Maniatis(Chapter 8). The oligonucleotide primers used were mainly based on thoseproposed by Marks et al. J. Mol. Biol (1991) 222, 581-597. The cDNA inthis case was prepared as described below. RNA (5 mg) was mixed in amicrocentrifuge tube with 10 μl 5× reverse transcriptase buffer [250 mMTris (pH8.3), 40 mM MgCl₂ and 50 mM DTT], 1 μl forward primer (25 μM),10 μl 1.25 mM dNTPs, 5 μl 10 mM DTT, 0.5 μl RNAguard (Pharmacia) towhich DEPC-treated H2O was added to obtain a volume of 50 μl. Thereaction mix was heated to 70° C. for 10 min and then cooled slowly to37° C., after which 100 u (0.5 μl) M-MLV reverse transcriptase(Pharmacia Cat. No. 27-0925-01) were added and the reaction incubated at37° C. for 1 h. The forward primer used for the generation of the lightchain cDNA was oligonucleotide CK2FOR (SEQ ID NO: 1) which is designedto hybridise to the CK constant region of murine kappa light chaingenes. For the heavy chain cDNA the forward primer CG1FOR (SEQ ID NO: 2)was used which hybridises to the CH1 constant domain of murine IgG 1.

7.3 Amino Acid Sequencing

The heavy and light chains of the 806.077 antibodies were isolated bySDS-PAGE and Western blotting and submitted for N-terminal amino acidsequencing. The results showed that the N-terminus of the light chainwas chemically blocked, however, sequence data was obtained for thefirst 34 N-terminal residues of the heavy chain (SEQ ID NO: 3). On thebasis of this amino acid sequence a specific DNA back primer wasdesigned for 806.077 heavy chain variable region PCR. This primer wascalled SP1back (SEQ ID NO: 7).

7.4 Isolation of Antibody Gene Fragments by PCR

Isolation of 806.077 heavy and light chain variable region genes wasperformed using the cDNA prepared as described above as template.General reaction conditions were as follows.

To 5 μl of the cDNA reaction was added 5 μl dNTPs (2.5 mM), 5 μl 10×Enzyme buffer (500 mM KCl, 100 mM Tris (pH8.3), 15 mM MgCl₂ and 0.1%gelatin), 1 μl of 25 pM/μl back primer, 1 μl of 25 pM/μl forward primer,0.5 μl thermostable DNA polymerase and DEPC-treated water to obtain avolume of 50 μl. The PCR conditions were set for 25 cycles at 94° C. for90 s; 55° C. for 60 s; 72° C. for 120 s, ending the last cycle with afurther 72° C. for 10 min incubation.

Using the general reaction conditions, the forward primer used for thegeneration of the light chain cDNA was oligonucleotide CK2FOR (SEQ IDNO: 1) and the for the heavy chain cDNA oligonucleotide CG1FOR (SEQ IDNO: 2). A number of reactions using a variety of different back primerswere performed for both the heavy and light chains to obtain desiredspecific PCR products.

In the case of the 806.077 light chain, on analysis a specific PCRproduct was obtained using the back primers VK1 back (SEQ ID NO: 4) andVK4back (SEQ ID NO: 5). Similarly specific PCR products were obtainedfor the heavy chain using VH1back (SEQ ID NO: 6) and SP1back primers(SEQ ID NO: 7). Reaction products were analysed on a 2% agarose gel.Products of the expected size, were excised and the DNA purified.

7.5 Cloning of the PCR Products into Bluescript KS+ Vector

For each antibody fragment, both the 5′ region (back primer)oligonucleotide and the 3′ region (forward primers) introduced arestriction site. The discrete PCR products were for both the VH and VKPCR reactions were therefore able to be cloned into the Bluescriptvector KS+ (Stratagene Cloning Systems) via the appropriate enzymerestriction sites using standard DNA manipulation methods (e.g. PCRproducts VH1back/CG1For was cloned via PstI/HindIII and VK4back/CK2Forvia SacI/HindIII). DNA was prepared from the clones obtained andrigorous sequencing of at least 12 clones of each construct performedusing automated fluorescent sequencing equipment (Applied Biosystems).The sequences were reviewed, compared and aligned using suitablecomputer software. Consensus sequences for both the VH and VK genes wereobtained and subsequently translated to their corresponding amino acidsequence.

The DNA and amino acid sequences obtained for the 806.077 light chainvariable (VK) region are described in SEQ ID NO: 8 and SEQ ID NO: 9respectively. The DNA and amino acid sequences obtained for the 806.077heavy chain variable (VH) region are described in SEQ ID NO: 10 and SEQID NO: 11 respectively. A clone containing the light chain wasdesignated VK4, and a clone containing the heavy chain sequnece wasdesignated VH14A.

EXAMPLE 8

Construction of Chimaeric Light Chain and Heavy Chain Fd Genes

The heavy and light chain genes which had been cloned into Bluescript(VK4 and VH14A in Example 7) were isolated by PCR using primers whichallowed specific amplification of only the variable region theappropriate genes but also introduced new unique enzyme restrictionsites. These restriction sites enabled the variable region genefragments to be cloned in frame with DNA fragments coding for both theappropriate antibody signal sequences and human constant regions. Thesignal and constant region sequences for the light and heavy chain Fdhad each been previously cloned into pNG3 and pNG4, derivatives of thepSG5 Eukaryotic plasmid expression vector.

The vector pNG3 was prepared as follows. Plasmid pSG5 (Stratagene, Cat.No. 216201) was digested with SalI and XbaI to remove the existing SV40promoter and polylinker sequence. A new polylinker was introduced by useof oligonucleotides SEQ NOS: 34 and 35 which were hybridised and clonedinto the SalI and XbaI cut pSG5 plasmid to give plasmid pNG1. The pNG1plasmid was cut with BgIII and HindIII and the BgIII-HindIII CMVpromoter fragment from pcDNA3 (Invitrogen, Cat. No. V790-20) cloned intothis site to give plasmid pNG2. Finally, the polyA region from pSG5 wasisolated by PCR as described in Example 7, section 7.4 but usingoligonucleotide sequences SEQ ID NOS: 36 and 37 with plasmid pSG5. ThePCR product was cut with XmaI and BamHI, purified by electrophoresis ona 2% agarose gel, isolated (e.g. with GENECLEAN, see example 7) thenligated into the XmaI-BamHI cut pNG2 plasmid to give pNG3.

The pNG4 vector was prepared as follows. The pNG3 vector was furthermodified such that the SacI restriction enzyme recognition site in thecloned CMV promoter fragment was corrupted by changing the DNA sequence.This was achieved by the use of a two step PCR mutagenesis reactionusing the pNG3 vector as a template. The PCR used-two complementaryoligonucleotide primers (SEQ ID NOS: 38 and 39) to mutate the Sac Irecognition sequence and 2 flanking primers (SEQ ID NOS: 40 and 41) forproduct amplification. Two Primer pairs (SEQ ID NOS: 38 and 41) and (SEQID NOS: 39 and 40) were used in a standard PCR reaction (as described inExample 7, section 7.4) to obtain the initial 2 PCR products, which wereisolated by electrophoresis on 2% agarose gels. Equimolar amounts ofeach product were mixed and reamplified using the flanking primers (SEQID NOS: 40 and 41) under the standard PCR reaction conditions to splicetogether and amplify the final PCR product. This product wassubsequently digested with the restriction enzymes NcoI and HindIII andcloned into the appropriately restricted and prepared pNG3 vector suchthat the mutated (SacI site minus) fragment replaced the original pNG3NcoI-Hind III (SacI site plus) fragment. This new vector was named pNG4.

A clone of the 806.077 murine light chain in the Bluescript KS+vector(VK4) was taken and amplified using the oligonucleotide primers 077VK-UP(SEQ ID NO: 12) and 077VK-DOWN (SEQ ID NO: 13). Similarly a 806.077heavy chain clone (VH14A) was amplified using 077VH-UP (SEQ ID NO: 14)and 077VH-DOWN (SEQ ID NO: 15). The PCR was performed as follows: To 100ng of plasmid DNA was added 5 μl dNTPs (2.5 mM), 5 μl 10×Enzyme buffer(see above), 1 μl of 25 pM/μl back primer, 1 μl of 25 pM/μl forwardprimer, 0.5 μl thermostable DNA polymerase and DEPC-treated water toobtain a volume of 50 μl. The PCR conditions were set for 15 cycles at94° C. for 90 s; 55° C. for 60 s; 72° C. for 120 s, ending the lastcycle with a further 72° C. for 10 min incubation. The products wereanalysed on a 2% agarose gel. The DNA was purified and the DNA fragmentdigested with the relevant restriction enymes in preparation forsubsequent vector cloning.

For secretion of antibody light chain, a double stranded DNA cassettewhich contained both the information for a Kozak recognition sequenceand a light chain signal sequence was designed. The cassette consistedof two individual oligonucleotides (SEQ ID NOS: 42 and 43) which werehybridised and subsequently cloned between cloned between the HindIIIand SacII restriction site of the pNG3 plasmid (which had beenappropriately restricted and isolated using standard methodology) tocreate the vector pNG3-Vkss. The DNA sequence of SEQ ID NO: 46, whichcontains the sequence for the human light chain kappa constant region,was digested with XmaI and XhoI and inserted between the XhoI and XmaIcut pNG-Vkss plasmid to give the vector pNG3-Vkss-HuCk (NCIMB no.40798). Furthermore, a neomycin resistance gene expression cassette wascloned into pNG3-Vkss-HuCk (from the pSG5 plasmid variant pSG5-Neovector, supplied from S. Green, Zeneca Pharmaceuticals; alternativesources include vectors such as pMC1neo, Stratagene cat. no. 213201).The neomycin resistance gene expression cassette was cloned as an XbaIfragment and cloned into the XbaI site of the pNG3-Vkss-HuCk and theorientation was checked using restriction enzyme digestion. This gaverise to the plasmid pNG3-Vkss-HuCk-Neo (NCIMB 40799). The light chaingene sequence described above was inserted, in frame, by cloningdirectly between the SacII and XhoI sites of the pNG3-Vkss HuCk-neovector. The PCR fragment obtained for the light chain gene was digestedwith SacII and XhoI restriction enzymes and cloned into the similarlyrestricted expression vector containing the VK signal and HuCK constantregion coding sequences. The chimaeric 806.077 light chain sequencecreated is shown in SEQ ID NOS: 16 and 17.

Similarly, for secretion of antibody heavy chain, a double stranded DNAcassette which contained both the information for a Kozak recognitionsequence and a heavy chain signal sequence was designed. The cassetteconsisted of two individual oligonucleotides (SEQ ID NOS: 44 and 45)which were hybridised and subsequently cloned between cloned between theHindIII and EcoRI restriction site of the pNG4 plasmid (which had beenappropriately restricted and isolated using standard methodology) tocreate the vector pNG4-VHss. Heavy chain gene sequences could thus beinserted. in frame, by cloning directly between the EcoRI and SacI sitesof the pNG4-VHss vector. The DNA sequence of SEQ ID NO: 47, whichcontains the coding sequence for human heavy chain IgG2CH1′ constantregion (SEQ ID NOS: 22 and 23) was digested with SacI and XmaI andcloned into pNG4-VHss cut with Sac and XmaI to give the vectorpNG4-VHss-HuIgG2CH1′ (NCIMB no. 40797). The PCR fragment obtained forthe heavy chain gene was digested with EcoRI and SacI restrictionenzymes and cloned into the similarly restricted expression vectorpNG4-VHss-HuIgG2CH1′ containing the VH signal and HuIgG2 CH1′ constantregion coding sequences. The chimaeric 806.077 HuIgG2 Fd chain sequencecreated is shown in SEQ ID NOS: 18 and 19.

In some instances it may be preferable to use other classes of chimaericheavy chain Fd constructs. To this end, variants of the heavy chainvector are made containing HuIgG1CH1′ (SEQ ID NOS: 20 and 21) orHuIgG3CH1′ (SEQ ID NOS: 24 and 25) which are substituted for theHuIgG2CH1′ (SEQ ID NOS: 22 and 23) gene. The sequences shown in SEQ IDNOS: 46 and 47 are prepared by a variety of methods including thosedescribed by Edwards (1987) Am. Biotech. Lab. 5, 38-44, Jayaraman et al.(1991) Proc. Natl. Acad. Sci. USA 88, 40844088, Foguet and Lubbert(1992) Biotechniques 13, 674-675 and Pierce (1994) Biotechniques 16,708. Preferably, the sequences shown in SEQ ID NOS: 46 and 47 areprepared by a PCR method similar to that described by Jayaraman et al.(1991) Proc. Natl. Acad. Sci. USA 88, 40844088.

Once the individual heavy and light chain sequences were constructed aheavy chain Fd gene expression cassette (including both promoter andgene was excised as a BgIII/SalI fragment and cloned between into theBamHI/SalI sites of the light chain vector to produce a co-expressionvector construct. This construct was transfected into NSO myeloma cells(ECACC No. 85110503) via standard techniques of electroporation andtransfectants selected for the property of G418 resistance, a traitwhich is carried as a selectable marker on the expression plasmidconstruct.

Alternatively the complete heavy chain Fd and light chain genes maysimply be excised from their respective vectors as HindIII/XmaIfragments and subsequently cloned into other expression vector systemsof choice.

EXAMPLE 9

Hybridization Test of Nucleic Acid Variations of Specific Nucleic AcidSequences

9.1 Hybridisation Test

A method for detecting variant nucleic acids containing sequencesrelated to specific 806.077 antibody sequences is described. Thesevariant nucleic acids may be present in a variety of forms such as theDNA from bacterial colonies or the DNA/RNA from eukaryotic cells fixedon to a membrane as described above in the screening of a cDNA libraryor as fragments of purified nucleic acid separated by gelelectrophoresis and then transfered to a suitable membrane as for thetechniques of Northern (Maniatis et al, Chapter 7, p39) or Southern(Maniatis, chapter 9, p31) hybridisation.

9.2 Hybridisation Probe

Hybridisation probes may be generated from any fragment of DNA or RNAencoding the specific 806.077 antibody nucleic sequence of interest,more specifically from the variable region, particularly the regionencoding CDR3 of this region. A synthetic oligonucleotide or itscomplementary sequence can be used as a specific probe for the CDR3encoding region.

A hybridisation probe can be generated from a synthetic oligonucleotideby addition of a radioactive 5′ phospate group from [γ-³² P]ATP by theaction of T4 polynucleotide kinase. 20 pmoles of the oligonucleotide areadded to a 20 μl reaction containing 100 mM Tris, pH7.5, 10 mM MgCl₂,0.1 mM spermidine, 20 mM dithiothreitol (DTT), 7.55 μM ATP, 55 μCi[γ-³²P]ATP and 2.5 u T4 polynucleotide kinase (Pharmacia BiotechnologyLtd, Uppsala, Sweden). The reaction is incubated for 30 minutes at 37°C. and then for 10 minutes at 70° C. prior to use in hybridisation.Methods for the generation of hybridisation probes from oligonucleotides(chapter 11) or from DNA and RNA fragments (chapter 10) are given inManiatis. A number of proprietary kits are also available for theseprocedures.

9.3 Hybridisation Conditions

Filters containing the nucleic acid are pre-hybridised in 100 ml of asolution containing 6×SSC, 0.1% SDS and 0.25% dried skimmed milk(Marvel™) at 65° C. for a minimum of 1 hour in a suitable enclosedvessel. A proprietary hybridisation apparatus such as model HB-1 (TechneLtd) provides reproducible conditions for the experiment.

The pre-hybridisation solution is then replaced by 10 ml of a probesolution containing 6×SSC, 0.1% SDS, 0.25% dried skimmed milk (e.g.Marvel™) and the oligonucleotide probe generated above. The filters areincubated in this solution for 5 minutes at 65° C. before allowing thetemperature to fall gradually to below 30° C. The probe solution is thendiscarded and the filters washed in 100 ml 6×SSC, 0.1% SDS at roomtemperature for 5 minutes. Further washes are then made in fresh batchesof the same solution at 30° C. and then in 10° C. increments up to 60°C. for 5 minutes per wash.

After washing, the filters are dried and used to expose an X-ray filmsuch as Hyperfilm™ MP (Amersham International) at −70° C. in alight-tight film cassette using a fast tungstate intensifying screen toenhance the photographic image. The film is exposed for a suitableperiod (normally overnight) before developing to reveal the photographicimage of the radio-active areas on the filters. Related nucleic acidsequences are identified by the presence of a photographic imagecompared to totally unrelated sequences which should not produce animage. Generally, related sequences will appear positive at the highestwash temperature (60° C.). However, related sequences may only showpositive at the lower wash temperatures (50, 40 or 30° C.).

These results will also depend upon the nature of the probe used. Longernucleic acid fragment probes will need to be hybridised for longerperiods at high temperature but may remain bound to related sequences athigher wash temperatures and/or at lower salt concentrations. Shorter,mixed or degenerate oligonucleotide probes may require less stringentwashing conditions such as lower temperatures and/or higherNa+concentrations. A discussion of the considerations for hybridisationprotocols is provided in Maniatis (chapter 11).

EXAMPLE 10

Pharmaceutical Compositions

The following illustrates representative pharmaceutical dosage formscontaining 806.077 antibody which may be used for therapy in combinationwith a suitable prodrug.

Injectable Solution for ADEPT

A sterile aqueous solution, for injection, containing per ml ofsolution:

806.077 antibody - CPG2 conjugate  1.0 mg Sodium acetate trihydrate  6.8mg Sodium chloride  7.2 mg Tween 20 0.05 mg

A typical dose of conjugate for adult humans is 30 mg followed 3 dayslater by three 1 g doses of prodrug administered at hourly intervals.Suitable CPG2 conjugates are any one of those conjugates described inExamples 105 and 106. Conjugates with HCPB may replace the CPG2conjugate in the table. Suitable HCPB conjugates are any one of thoseconjugates described in Examples 48-101.

Injectable Solution for Tumour Immunotherapy

A sterile aqueous solution, for injection, containing per ml ofsolution:

806.077 antibody - B7 conjugate  1.0 mg Sodium acetate trihydrate  6.8mg Sodium chloride  7.2 mg Tween 20 0.05 mg

A typical dose of conjugate for adult humans is 30 mg. A suitableconjugate is described in Example 104.

EXAMPLE 11

Construction of Initial 806.077 Humanised Antibody Heavy and Light ChainVariable Region Genes

Firstly an overview of the humanisation strategy is set out in thefollowing text. The purpose of antibody humanisation is to combine thebinding site of a non-human antibody into the supporting framework of ahuman antibody while maintaining the characteristic antigen bindingaffinity and specificity properties of the parent antibody. Thefeasibility of such antibody engineering is a consequence of the closesequence and structural homology of immunoglobulins from differentmammalian species.

In its most basic form the approach involes the transfer of the sixhypervariable regions or complementarity determing regions (CDRs) fromone antibody Fv region to another, as first described in Jones etalNature (1986) 321 522-525. However, experience has shown that inaddition to the CDRs it is often necessary that amino acids in theantibody framework also need to be transferred for the process to besuccessful since such residues sometimes appear to contact and influencethe conformation of the CDR loops.

In the case of the 806.077 antibody an “Initial” humanised version ofthe antibody was made which comprises the six murine CDRs and a numberof framework residue substitutions. This construct was used as atemplate from which further variants (Examples 1247) were made byintroducing additional “murine” residue substitutions. The rest of thisExample describes the Initial humanised construct in detail.

The human antibody heavy chain variable region NEWM (Poljak, R. J et al(1974) PNAS 71 3440-3444) and the light chain kappa variable region REI(Palm, W and Hilschmann, N. Z. (1975) Physiol. Chem. 356 167-191 werechosen to form the acceptor human antibody framework. Numerous examplesof successful humanisations-using this Fv framework have been describedin the literature and the 3 dimensional structure of these two proteindomains has been solved. Based on comparison of the murine 806.077 heavyand light chain variable region protein sequences with their closestrelated Kabat murine subgroup concensus sequences (and the individualsequence members) and the human NEWM and REI protein sequences,individual DNA sequences were designed to encode for the Initialhumanised antibody which incorporated the murine CDRs and any additionalframework substitutions considered to be of importance.

The murine 806.077 CDR sequences incorporated are described in SEQ IDNOs: 26; 27 and 28 for the light chain variable region and are found atpositions 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) repectively. TheCDRs incorporated in the heavy chain variable region are described inSEQ ID NOs: 29,31 and 32 being at positions 31-35 (CDR1), 50-65 (CDR2),95-102 (CDR3) respectively (using Kabat nomenclature). In the heavychain variable region the additional changes V24A; S27F; T28N; F29I;S30K;.V71A; A92H; R93V (Kabat nomenclature) were made to the NEWMframework and in the light chain variable region no additional frameworkchanges were made.

Individual synthetic DNA sequences were designed to encode for theinitial version of the 806.077 humanised antibody heavy (806.077HuVH1)and light chain (806.077HuVK1) variable regions in which the CDRs andany additional framework residue changes were incorporated. The antibodyvariable gene sequences shown in SEQ ID NOS: 48 and 53 may be preparedby a variety of methods including those described by Edwards (1987) Am.Biotech. Lab. 5, 38-44, Jayaraman et al. (1991) Proc. Natl. Acad. Sci.USA 88, 40844088, Foguet and Lubbert (1992) Biotechniques 1, 674-675 andPierce (1994) Biotechniques 16, 708. Preferably, the DNA sequences shownin SEQ ID NOS: 48 and 53 are prepared by a PCR method similar to thatdescribed by Jayaraman et al. (1991) Proc. Natl. Acad. Sci. USA 88,4084-4088.

The humanised 806.077 antibody variable light chain gene sequence (SEQID NO: 49 and 50) was inserted, in frame, by cloning into thepNG3-Vkss-HuCK-Neo (NCIMB no. 40799) expression vector. To achieve this,the synthetic PCR DNA fragment encoding the humanised variable lightchain gene (SEQ ID NO: 48) was digested with SacII and XhoI restrictionenzymes and cloned into the similarly restricted pNG3-Vkss-HuCK-Neovector which contained the VK signal sequence and HuCK constant regioncoding sequences. The DNA and protein sequence of completed humanised806.077 light chain sequence (806.077HuVK1-HuCK) produced, together withits signal sequence, are shown in SEQ ID NOS: 51 and 52 respectively andthe vector named pNG3-Vkss-806.077HuVK1-HuCK-Neo.

Similarly, for the humanised antibody heavy chain, the humanisedvariable heavy chain gene sequence SEQ ID NO: 54 and 55 was inserted, inframe, by cloning directly into the pNG4-VHss-HulgG2CH1′ (NCIMB no.40797) expression vector. To achieve this, the synthetic PCR fragmentobtained for the humanised heavy chain gene (SEQ ID NOS: 53) wasdigested with EcoRI and SacI restriction enzymes and cloned into thesimilarly restricted pNG4-VHss-HuIgG2CH1′ vector which contained the VHsignal sequence and HuIgG2 CH1′ constant region coding sequences. TheDNA and protein sequence of completed humanised 806.077 Fd heavy chainsequence (806.077HuVH1-HulgG2 Fd) produced, together with its signalsequence, are shown in SEQ ID NOS: 56 and 57 respectively and the vectornamed pNG4-VHss-806.077HuVH1-HuIgG2 CH1′.

The Initial humanised antibody construct was produced by constructing aco-expression plasmid containing both 806.077 HuVK1 light chain and806.077 HuVH1 heavy chain variable region antibody genes. The plasmidpNG3-Vkss-806.077HuVK1-HuCK-Neo vector, which contains the humanisedlight chain variable region HuVK1 (SEQ ID NOS: 49 and 50) was digestedusing the restriction enzymes BamHI and SalI and the vector run on a 1%agarose gel, the vector band was excised and purified. The plasmidpNG4-VHss-806.077HuVH1-HuIgG2 CH1′ (which contains the humanised806.077HuVH1 heavy chain variable region (SEQ ID NOS: 56 and 57)) wasdigested using the restriction enzymes BgIII and SalI, the reaction runon a 2% agarose and the fragment band excised and purified. The DNAfragment recovered was subsequently ligated into the preparedpNG3-Vkss-806.077HuVK1-HuCK-Neo vector to produce clones of the desiredHuVH1/HuVK1 co-expression vector.

These constructs were transfected into NSO myeloma cells (ECACC No.85110503) via standard techniques of electroporation and transfectantsselected for the property of G41 8 antibiotic resistance. The clonesobtained were tested for both antibody expression in the anti-humanantibody Fd ELISA and CEA binding ELISA assays described below.

For the CEA ELISA each well of a 96 well immunoplate (NUNC MAXISORB™)was coated with 50 ng CEA in 50 mM carbonate/bicarbonate coating bufferpH9.6 (buffer capsules—Sigma C3041) and incubated at 4° C. overnight.The plate was washed three times with PBS+0.05% Tween 20 and thenblocked 150 μl per well of 1% BSA in PBS +0.05% Tween 20 for 1 hour atroom temperature. The plate was washed as previously described, 100 μlof test sample added per well and incubated at room temperature for 2hours. Again the plate was washed three times with PBS+0.05% Tween 20,100 μl per well of a 11500 dilution of HRPO-labelled goat anti-humankappa antibody (Sigma A 7164) was added, in 1% BSA in PBS-Tween 20 andincubated at room temperature on a rocking platform for at least 1 hour.The plate was washed as before and then once more with PBS. To detectbinding add 100 μl per well developing solution (one capsule ofphosphate-citrate buffer—Sigma P4922-dissolved in 100 mls H₂O to whichis added one 30 mg tablet o-phenylenediamine dihydrochloride—SigmaP8412) and incubated for up to 15 minutes. The reaction was stopped byadding 75 μl 2M H₂SO₄, and absorbance read at 490 nm.

In the anti-human antibody Fd ELISA, each well of a 96 well immunoplatewas coated with 1.2 μg sheep anti-human Fd antibody (Binding Site PC075)in 50 mM carbonate/-bicarbonate coating buffer pH9.6 (buffercapsules—Sigma C3041) and incubated at 4° C. overnight. The plate waswashed three times with PBS+0.05% Tween 20 and then blocked with 150 μlper well of 1% BSA in PBS+0.05% Tween 20 for 1 hour at room temperature.The plate was washed as previously described, 100 μl of test sampleadded per well and incubated at room temperature for 2 hours. Again theplate was washed three times with PBS+0.05% Tween 20, 1 001l per well ofHRPO-labelled goat anti-human kappa antibody (Sigma A 7164) was added in1% BSA in PBS-Tween 20 and incubated at room temperature on a rockingplatform for at least 1 hour. Wash plate as before and then once morewith PBS. To detect binding, developing solution etc was added asdescribed above for the CEA binding assay.

The clones found to show the best expression and CEA binding levels wereselected for further expansion into 24 well plates and re-tested. Thebest clone according to these assay criteria was selected and expandedsuch that a one liter production was undertaken , which was seeded usinga 1:10 dilution of a confluently grown culture (i.e. 100 mls into 900mls of fresh culture medium) and the grown for a further 14 days. Thehuman F(ab′)₂ antibody fragment was then purified from the culturesupernatant as described in Example 102.

EXAMPLES 12-38

Further Combinations of Humanised Heavy and Light Chain Variable RegionGene Variants: Construction of 806.077 Humanised Heavy and LightVariable Region Variants.

The Initial humanised 806.077 variable region genes were also used forthe subsequent construction of further gene constructs which containedadditional murine framework residues. Modifications of the genesequences were achieved (in the majority of cases) by cassettemutagenesis. In this technique part of the original gene was removed viarestriction with two appropriate unique enzymes from the completeplasmid vector and then replaced by a double stranded DNA cassette(consisting of two complementary oligonucleotides hybridised together toform a DNA fragment with the appropriate cohesive ends) by directligation into the prepared plasmid thus reconstituting the gene but nowcontaining desired DNA changes. Further combinations of mutations withineither the heavy or light chain could be also be produced by simple DNAfragment exchanges between the appropriate variants by utilising theavailable unique restriction enzyme sites. Three further variants of thehumanised light chain variable region were produced in addition to theoriginal sequence HuVK1 (SEQ ID NO: 49 and 50) and these were calledHuVK2, HuVK3 and HuVK4 repectively. The light chain variable regionvariant HuVK2 was a modification of the original HuVK1 coding sequencein order to produce the amino acid change M4L (Kabat nomenclature), withthe gene (SEQ ID NO: 49) being mutated by cassette mutagenesis. Theplasmid pNG3-Vkss-806.077HuVK1-HuCK-Neo (which contains the completehumanised light chain (SEQ ID NOS: 49 and 50) was digested using therestriction enzymes SacII and NheI. The digest was then loaded on a 2%agarose gel and the excised fragment separated from the remainingvector. The vector DNA was then excised from the gel, recovered andstored at −20° C .until required. Two oligonucleotides (containing thedesired base changes) were designed and synthesised (SEQ ID NO: 58 and59). These two oligonucleotides were hybridised by adding 200 pmoles ofeach oligonucleotide into a total of 30 μl of H₂O, heating to 95° C. andallowing the solution to cool slowly to 30° C. 100 pmoles of theannealed DNA product was then ligated directly into the previouslyprepared vector. This DNA “cassette” exchange produced the desired HuVK2DNA and protein sequence (SEQ ID NO: 60 and 61) already in place in theexpression vector pNG3-Vkss-806.077HuVK2-HuCK-Neo.

Similarly, HuVK3 with the amino acid changes D1Q; Q3V; M4L (Kabatnomenclature) was constructed using synthetic oligonucleotides (SEQ IDNO: 62 and 63) to produce the desired HuVK3 DNA and protein sequence(SEQ ID NO: 64 and 65) again already in place in the expression vectorpNG3-Vkss-806.077HuVK3-HuCK-Neo. The light chain variable region variantHuVK4 was produced by a different technique, as there were not uniquerestriction enzyme sites available close to the mutation site. HuVK4,with the amino acid change L47W, was produced by a PCR mutagenesistechnique. The vector pNG3-806.077HuVK1-HuVK-Neo was used as thetemplate for two PCR reactions (94° C., 90 sec; 55° C., 60 sec; 72° C.,120 sec for 15 cycles, all buffers, etc., as previously described).Reaction A used the synthetic oligonucleotide sequence primers SEQ IDNOS: 66 and 67 and reaction B the synthetic oligonucleotide sequenceprimers SEQ ID NOS: 68 and 69. The products of these PCR reactions (Aand B) were fragments of length 535 base pairs and 205 base pairsrespectively. These reaction products were run on a 2% agarose gel andseparated from any background products. Bands of the expected size wereexcised from the gel and recovered. Mixtures of varying amounts of theproducts A and B were made and PCR reactions performed using thesynthetic oligonucleotides SEQ ID NOS: 66 and 68. The resulting product(ca.700 base pairs) was digested with the restriction enzymes SacII andXhoI and the cleavage products separated on a 2% agarose gel. The bandof the expected 310 base pairs size was excised from the gel andrecovered. This fragment was then ligated into the vectorpNG3-806.077HuVK1-HuVK-Neo vector (which had been previously cut withthe restriction enzymes SacII/XhoI and subsequently isolated) and thuscreated the desired HuVK4 DNA and protein sequence (SEQ ID NO: 70 and71) within the expression vector pNG3-Vkss-806.077HuVK4-HuCK-Neo.

Six further variants of the humanised heavy chain variable region wereproduced in addition to the original HuVH1 sequence (SEQ ID NO: 54 and55) and these were called HuVH2 to HuVK7 respectively. The heavy chainvariable region variant HuVH2 was a modification of the original HuVH1coding sequence in order to produce the amino acid change G49A (Kabatnomenclature), with the gene (SEQ ID NO: 54) being mutated by cassettemutagenesis. The plasmid pNG4-VHss-806.077HuVH1-HuIgG2 CH1′ (whichcontains the complete humanised, IgG2 heavy chain Fd (SEQ ID NOS: 56 and57) was digested using the restriction enzymes StuI and NotI. The digestwas then loaded on a 2% agarose gel and the excised fragment separatedfrom the remaining vector. The vector DNA was then excised from the gel,recovered and stored at −20° C. until required. Two oligonucleotideswere designed, synthesised (SEQ ID NO: 72 and 73), hybridised and theproduct ligated directly into the previously prepared vector. This DNA“cassette” exchange produced the desired HuVH2 DNA and protein sequence(SEQ ID NO: 74 and 75) already in place in the expression vectorpNG4-VHss-806.077HuVH2-HuIgG2 CH1′.

Similarly, HuVH3 with the amino acid changes T73S; F78A (Kabatnomenclature) was constructed using synthetic oligonucleotides (SEQ IDNO: 76 and 77), however. in this case, the vectorpNG4-VHss-806.077HuVH1-HuIgG2 CH1′ was digested using the restrictionenzymes NotI and SacII. The synthetic DNA cassette was ligated directlyinto the previously prepared vector to produce the desired HuVH3 DNA andprotein sequence (SEQ ID NO: 78 and 79) in the expression vectorpNG4-VHss-806.077HuVH3-HuIgG2 CH1′.

HuVH4 with the amino acid changes G49A; T73S; and F78A (Kabatnomenclature) combines the HuVH2 (SEQ ID NO: 74 and 75) and HuVH3 (SEQID NO: 78 and 79) variants. This was achieved by digesting thepNG4-VHss-806.077HuVH3-HuIgG2 CH1′ vector with the enzymes NotI and NheIand isolating the ca. 200 base pairs NotI/NheI restriction fragmentafter separation on a 2% agarose gel. The fragment was recovered andsubsequently ligated into the pNG4-VHss-806.077HuVH2-HuIgG2 CH1′ vector(which had been digested with the same Not I and NheI restrictionenzymes and the vector fragment purified). The resulting clonescontained the desired HuVH4 DNA and protein sequence ((SEQ ID NO: 80 and81) in the expression vector pNG4-VHss-806.077HuVH4-HuIgG2 CH1′.

HuVH5 with the amino acid changes V67A (Kabat nomenclature) wasconstructed using synthetic oligonucleotides (SEQ ID NO: 82 and 83).Again, the vector pNG4-VHss-806.077HuVH1-HuIgG2 CH1′ was digested usingthe restriction enzymes NotI and SacII. The synthetic DNA cassette wasligated directly into the previously prepared vector to produce thedesired HuVH5 DNA and protein sequence (SEQ ID NO: 84 and 85) in theexpression vector pNG4-VHss-806.077HuVH5-HuIgG2 CH1′.

HuVH6 with the amino acid changes V67A;T73S and F78A (Kabatnomenclature) was constructed using synthetic oligonucleotides (SEQ IDNO: 86 and 87) and for this mutant the vectorpNG4-VHss-806.077HuVH1-HuIgG2 CH1′ was digested using the restrictionenzymes NotI and SacII. The synthetic DNA cassette was ligated directlyinto the previously prepared vector to produce the desired HuVH6 DNA andprotein sequence (SEQ ID NO: 88 and 89) in the expression vectorpNG4-VHss-806.077HuVH6-HuIgG2 CH1′.

HuVH7 with the amino acid changes G49A; V69A; T73S; and F78A (Kabatnomenclature) combines the HuVH2 (SEQ ID NO: 74 and 75) and HuVH6 (SEQID NO: 88 and 89) variants. This was achieved by digesting thepNG4-VHss-806.077HuVH6-HuIgG2 CH1′ vector with the enzymes NotI and NheIand isolating the ca. 200 base pairs NotI/NheI restriction fragmentafter separation on a 2% agarose gel. The fragment was recovered andligated into the pNG4-VHss-806.077HuVH2-HuIgG2 CH1′ vector (which hadbeen digested with the same Not I and NheI restriction enzymes and thevector fragment purified). The resulting clones contained the desiredHuVH7 DNA and protein sequence (SEQ ID NO: 90 and 91) in the expressionvector pNG4-VHss-806.077HuVH7-HuIgG2 CH1′.

Combinations of such humanised heavy and light chain variable genevariants were made by excising the heavy chain Fd gene variantexpression cassette (including both promoter and gene excised as aBglII/SalI fragment) and cloning this fragment into the BamHI/SalI sitesof the light chain variant vector to produce a co-expression vectorconstruct. A listing of the possible combinantions of variants based onthe humanised heavy and light chain variants previously described isshown in the table below.

TABLE Combinations of humanised heavy and light chain variable regionvariants. Heavy chain Light chain Example variable SEQ ID variable SEQID No. region NOS: region NOS: Co-expression Plasmid Vector 11 HuVH1 54and 55 HuVK1 49 and 50 pNG 806HuVH1/HuVK1/HulgG2 12 HuVH1 54 and 55HuVK2 60 and 61 pNG 806HuVH1/HuVK2/HuIgG2 13 HuVH1 54 and 55 HuVK3 64and 65 pNG 806HuVH1/HuVK3/HuIgG2 14 HuVH1 54 and 55 HuVK4 70 and 71 pNG806HuVH1/HuVK4/HulgG2 15 HuVH2 74 and 75 HuVK1 49 and 50 pNG806HuVH2(HuVK1/HuIgG2 16 HuVH2 74 and 75 HuVK2 60 and 61 pNG806HuVH2IHuVK2/HuIgG2 17 HuVH2 74 and 75 HuVK3 64 and 65 pNG806HuVH2/HuVK3/HuIgG2 I8 HuVH2 74 and 75 HuVK4 70 and 71 pNG806HuVH2(HuVK4/HuIgG2 19 HuVH3 78 and 79 HuVK1 49 and 50 pNG806HuVH3/HuVK1/HuIgG2 20 HuVH3 78 and 79 HuVK2 60 and 61 pNG806HuVH3/HuVK2/HuIgG2 21 HuVH3 78 and 79 HuVK3 64 and 65 pNG806HuVH3/HuVK3/HuIgG2 22 HuVH3 78 and 79 HuVK4 70 and 71 pNG806HuVH3/HuVK4/Hu1gG2 23 HuVH4 80 and 81 HuVK1 49 and 50 pNG806HuVH4/HUVK1/HuIgG2 24 HuVH4 80 and 81 HuVK2 60 and 61 pNG806HuVH4/HuVK2/HuIgG2 25 HuVH4 80 and 81 HuVK3 64 and 65 pNG806HuVH4/HuVK3/HuIgG2 26 HuVH4 80 and 81 HuVK4 70 and 71 pNG806HuVH4/HuVK4/HuIgG2 27 HuVH5 84 and 85 HuVK1 49 and 50 pNG806RuVH5/HuVK1/HuIgG2 28 HuVH5 84 and 85 HuVK2 60 and 61 pNG806HuVH5/HuVK2/HuIgG2 29 HuVH5 84 and 85 HuVK3 64 and 65 pNG806HuVH5/HuVK3/HuIgG2 30 HuVH5 84 and 85 HuVK4 70 and 71 pNG806HuVH5/HuVK4/HuIgG2 31 HuVH6 88 and 89 HuVK1 49 and 50 pNG806HuVH6/HuVK1/HuIgG2 32 HuVH6 88 and 89 HuVK2 60 and 61 pNG806HuVH6/HuVK2/HulgG2 33 HuVH6 88 and 89 HuVK3 64 and 65 pNG806HuVH6/HuVK3/HuIgG2 34 HuVH6 88 and 89 HuVK4 70 and 71 pNG806HuVH6/HuVK4/HuIgG2 35 HuVH7 90 and 91 HuVK1 49 and 50 pNG806RuVH7/HuVK1/HuIgG2 36 HuVH7 90 and 91 HuVK2 60 and 61 pNG806HuVH7/HuVK2/HuIgG2 37 HuVH7 90 and 91 HuVK3 64 and 65 pNG806HuVH7/HuVK3/HuIgG2 38 HuVH7 90 and 91 HuVK4 70 and 71 pNG806HuVH7/HuVK4/HuIgG2

Analogously with Example 11, Example 14 was produced by constructing aco-expression plasmid containing both the 806.077 HuVK4 light chain andthe 806.077 HuVH1 heavy chain variable region antibody genes. In thiscase the plasmid the pNG3-Vkss-806.077HuVK4-HuCK-Neo vector, whichcontains the humanised light chain variable region HuVK1 (SEQ ID NOS: 70and 71) was digested using the restriction enzymes BamHI and SalI andthe vector run on an 1% agarose gel and the vector band purified. Theplasmid pNG4-VHss-806.077HuVH1-HuIgG2 CH1′ (which contains the humanised806;077 HuVH1 heavy chain variable region (SEQ ID NOS: 56 and 57) wasdigested using the restriction enzymes BglII and SalI, the reaction runon an 2% agarose and the fragment band excised and purified. The DNAfragment recovered was ligated into the preparedpNG3-Vkss-806.077HuVK4-HuCK-Neo vector to produce clones of the desiredHuVH1/HuVK4 co-expression vector.

As described in Example 11, these constructs were transfected into NSOmyeloma cells (ECACC No. 85110503) via standard techniques ofelectroporation and transfectants selected for the property of G418resistance. The clones obtained were tested for both antibody expressionin anti-human antibody Fd ELISA and CEA binding ELISA assays. Clonesfound to show the best expression and CEA binding levels were selected,expanded and product expressed. Human F(ab′)₂ antibody fragment was thenpurified from the culture supernatant as described in Example 102.

EXAMPLE 39-47

Expression of Humanised F(ab′)₂ Fragments With Various Classes of HumanHeavy Chain Constant Regions

Other classes of chimaeric heavy chain Fd constructs may be used.Accordingly, additional variants of the heavy chain vectors have beenmade which contain either HuIgG1CH1′ (SEQ ID NOS: 20 and 21) orHuIgG3CH1′ (SEQ ID NOS: 24 and 115), the constant regions of which aresubstituted for the HuIgG2CH1′ gene (SEQ ID NOS: 22 and 23). The vectorscreated were pNG4-VHss-HuIgG1 CH1′ and pNG4-VHss-HuIgG3 CH1′respectively. The heavy. chain antibody variable region in question canbe excised from the appropriate pNG4-VHss-“VH variable region”-HuIgG2CH1′ plasmid by digestion with EcoRI and SacI restriction enzymes andcloned into the similarly restricted pNG4-VHss-HuIgG1CH1′ orpNG4-VHss-HuIgG3 CH1′ vector and thus produce a completed heavy chain Fdsequence. As described above, once the individual heavy and light chainsequences are constructed, a heavy chain Fd gene expression cassette(including both promoter and gene can be excised by restrictiondigestion and the fragment cloned between into the appropriate sites ofthe light chain vector to produce the final co-expression vector. Thetable below describes Examples 39-47 in which various heavy and lightchain variable regions have been combined with a number of differentclasses of human heavy chain constant regions.

In Example 44, the vector pNG4-VHss-HuIgG3 CH1′ was digested with therestriction enzymes EcoRI and SacI restriction enzymes and the vectorfragment isolated as previously described. The HuVH1 heavy chainantibody variable region (SEQ ID NOS: 54 and 55) was excised from thepNG4-VHss-806.077HuVH1-HuIgG2 CH1′ plasmid by digestion with EcoRI andSacI restriction enzymes and the fragment cloned into the similarlyrestricted pNG4-VHss-HuIgG3 CH1′ vector to produce a completed humanisedIgG3 heavy chain Fd sequence (SEQ ID NOS: 94 and 95) in the completedvector pNG4-VHss-806.077HuVH1-HuIgG3 CH1′. The heavy chain Fd geneexpression cassette (including both promoter and gene) was excised as aBglII/SalI fragment and cloned into the BamHI/SalI sites of the lightchain vector pNG3-Vkss-806.077HuVK1-HuCK-Neo (containing the HuVK1-HuCKhumanised light chain SEQ ID NOS: 51 and 52) which had been digestedusing the restriction enzymes BamHI and SalI, run on an 1% agarose, thevector band purified. This produced a co-expression vector (pNG806HuVH1/HuVK3/HuIgG3) from which the humanised806.077HuVH1/HuVK1-HuIgG3/Kappa.Fd antibody fragment could be expressed.

TABLE SEQ Example Humanised ID Humanised SEQ ID No. heavy chain NOSlight chain NOS Co-expression Plasmid Vector 39 HuVH1-HuIgG1 92 andHuVK1-HuCK 51 and 52 Png 806HuVH1/HuVK1/HuIgG1 93 40 HuVH1-HuIgG2 56 andHuVK1-HuCK 51 and 52 pNG 806HuVH1/HuVK1/HuIgG2 57 41 HuVH1-HuIgG3 94 andHuVK1-HuCK 51 and 52 pNG 806HuVH1/HuVK1/HuIgG3 95 42 HuVH1-HuIgG1 92 andHuVK3-HuCK 96 and 97 pNG 806HuVH1/HuVK3/HuIgG1 93 43 HuVH1-HuIgG2 56 andHuVK3-HuCK 96 and 97 pNG 806HuVH1/HuVK3/HuIgG2 57 44 HuVH1-HuIgG3 94 andHuVK3-HuCK 96 and 97 pNG 806HuVH1/HuVK3/HuIgG3 95 45 HuVH1-HuIgG1 92 andHuVK4-HuCK 98 and 99 pNG 806HuVH1/HuVK4/HuIgG1 93 46 HuVH1-HuIgG2 56 andHuVK4-HuCK 98 and 99 pNG 806HuVH1/HuVK4/HuIgG2 57 47 HuVH1-HuIgG3 94 andHuVK4-HuCK 98 and 99 pNG 806HuVH1/HuVK4/HuIgG3 95

In Example 47, the vector pNG4-VHss-HuIgG3 CH1′ was digested with EcoRIand SacI restriction enzymes and the vector fragment isolated. The HuVH1heavy chain antibody variable region (SEQ ID NOS: 54 and 55) was excisedfrom the pNG4-VHss-HuVH1-HuIgG2 CH1′ plasmid by digestion with EcoRI andSacI restriction enzymes and the fragment cloned into the similarlyrestricted pNG4-VHss-HuIgG3 CH1′ vector. This produces a completedhumanised IgG3 heavy chain Fd sequence (SEQ ID NOS: 94 and 95) in thecompleted vector pNG4-VHss-806.077HuVH1-HuIgG3 CH1′. The heavy chain Fdgene expression cassette (including both promoter and gene) was excisedas a BglII/SalI fragment and cloned into the BamHI/SalI sites of thelight chain vector pNG3-Vkss-806.077HuVK4-HuCK-Neo vector, (containingHuVK4-HuCK humanised light chain SEQ ID NOS: 98 and 99 ) which had beendigested using the restriction enzymes BamHI and SalI, run on an 1%agarose, the vector band purified. This produced a co-expression vectorconstruct pNG 806HuVH1/HuVK4/HuIgG3 from which the humanisedHuVH1/HuVK1-HuIgG3/Kappa.Fd antibody fragment could be expressed.

The other Examples shown in the table above were all produced in asimilar manner to that described in the Examples 44 and 47. However, inthe case of the constructs containing human IgG1, the finalco-expression vector construction was made by cloning the heavy chain Fdgene expression cassette (including both promoter and gene) excised as aBglII/BamHI fragment (because there is an internal SalI restriction sitein the HuIgG1 CH1′ constant region gene) and cloned into the BamHI siteof the appropriately prepared light chain vector. In this case theorientation of the heavy chain cassette must be checked. This wasachieved by restriction digestion (e.g. with the restriction enzyme HindIII) and agarose gel electrophoresis analysis in which the resultingfragment sizes were viewed relative to comparable fragments from asimilarly digested HuIgG2 version (Examples 11-38). When thefragmentation patterns matched for both constructs we could be sure thatthe heavy chain cassette was in the correct orientation.

As previously described in Example 1, these constructs were transfectedinto NSO myeloma cells (ECACC No. 85110503) via standard techniques ofelectroporation and transfectants selected for the property of G418resistance. The clones obtained were tested for both antibody expressionin the anti-human antibody Fd ELISA and CEA binding ELISA assays and theclones found to show the best expression and CEA binding levels wereselected, expanded and grown for gene expression. As before, the humanF(ab′)₂ antibody fragment was then purified from the culture supernatantas described in Example 102.

EXAMPLE 48

Preparation of Humanised 806.077 F(ab′)₂-[A248S,G251T,D253K]HCPB FusionProtein

This Example describes the preparation of a gene encoding a humanised Fdheavy chain fragment of 806.077 linked to [A248S,G251T,D253K]HCPB andits co-expression with a gene encoding a humanised light chain of806.077 and a gene encoding the pro domain of human carboxypeptidase Bto give the F(ab′)₂ protein with a molecule of [A248S,G251T,D253K]HCPBat the C-terminus of each of the heavy chain fragments. The constant andhinge regions of of the humanised Fd heavy chain fragment are derivedfrom the human IgG3 antibody isotype. The expressed protein is alsoreferred to as antibody-enzyme fusion protein.

(a) Preparation of a Gene Encoding Humanised Fd Heavy Chain Fragment of806.077 Linked to [A248S, G251 T, D253K]HCPB and its Cloning into pEE6

A gene encoding humanised 806.077 Fd linked to [A248S,G251T,D253K]HCPBwas generated by PCR from pZEN 1921 (Reference Example 2). A first PCRwas set up with template pZEN1921 (2 ng) and oligonucleotides SEQ ID NO:100 and SEQ ID NO: 101 (100 pM of each) in buffer (100 μl) containing 10mM Tris-HCl (pH8.3), 50 mM KCL, 1.5 mM MgCl₂, 0.125 mM each of dATP,dCTP, dGTP and dTTP. The reaction was incubated at 94° C. for 5 min thenthermostable DNA polymerase (2.5 u, 0.5 μl) was added and the mixtureoverlaid with mineral oil (100 μl) and the reaction mixture incubated at94° C. for 1 min, 53° C. for l min and 72° C. for 2.5 min for 25 cycles,plus 10 min at 72° C. The PCR product of 536 base pairs was isolated byelectrophoresis on a 1% agarose (Agarose type 1, Sigma A-6013) gelfollowed by excision of the band from the gel and isolation of the DNAfragment.

A second PCR was set up with template IgG3-pBSIIKS+(8.7 ng, described inReference Example 4) and oligonucleotides SEQ ID NO: 102 and SEQ ID NO:103 and the 954 base pairs fragment isolated as described above. Theproducts from the 2 PCRs were combined (either at 0.2, 1.0 or 5.0 ng/μl)in PCR buffer as described above. The mixture was incubated for at 94°C. for 5 min then 10 cycles at 94° C. for 1 min and 63° C. for 4 min.Oligos SEQ ID NOS: 101 and 102 (100 pM of each) in PCR buffer (50 μl)were added. After incubation at 94° C. for 3 min, the mixture wasfurther incubated at 94° C. for 1.5 min, 53° C. for 2 min and 72° C. for2 min for 25 cycles plus 10 min at 72° C. In this process, the G base atposition 508 in SEQ ID NO: 115 was changed to an A base.

The PCR product of 1434 base pairs was isolated by electrophoresis on a1% agarose gel, purified and digested with NheI (20 u) and XbaI (80 u)(New England Biolabs Inc.,) in a total volume of 100 μl containing 10 mMTris HCl (pH7.9), 50 mM NaCl, 10 mM MgCl₂, 1 mM DTT and BSA (100 g/ml)for 4 h at 37° C. The resulting fragment was again isolated byelectrophoresis on a 1% agarose gel and purified. In a similardigestion, vector pNG4-VHss-806.077huVH1-HuIgG2CH1′ (10 μg; Example 11)was cut with NheI and XbaI then calf intestinal alkaline phosphatase (1μl; New England Biolabs, 10 u/μl) was added to the digested plasmid toremove 5′ phosphate groups and incubation continued at 37° C. for afurther 30 minutes. Phosphatase activity was destroyed by incubation at70° C. for 10 minutes. The NheI-XbaI cut plasmid was purified from anagarose gel. The NheI-XbaI digested PCR product from above (about 500ng) was ligated with the above cut plasmid DNA (about 200 ng) in 20 μlof a solution containing 50 mM Tris-HCl (pH7.8), 10 mM MgCl₂, 10 mM DTT,1 mM ATP, 50 μg/ml BSA and 400 u T4 DNA ligase (New England Biolabs,Inc) at 25° C. for 4 h. A 1 μl aliquot of the reaction was used totransform 20 μl of competent E. coli DH5α cells. Transformed cells wereplated onto L-agar plus 100 g/ml ampicillin. Potential clones containingthe gene for humanised 806.077 Fd-[A248S,G251T,D253K]HCPB wereidentified by PCR. Each clone was subjected to PCR as described abovewith oligonucleotides SEQ ID NOS: 104 and 105. A sample (10 μl) of thePCR reaction was analysed by electrophoresis on a 1% agarose gel. Clonescontaining the required gene were identified by the presence of a 512base pairs PCR product. Clones producing the 512 base pairs band wereused for DNA minipreps. The DNA samples were checked by digestion withHindIII and XbaI for the presence of 3751 base pairs and 1862 base pairsfragments. Clones containing these fragments on digestion of the DNAwith HindIII and XbaI were used for large scale plasmid DNA preparationand the sequence of the insert confirmed by DNA sequence analysis. Thesequence of the expected insert is shown in SEQ ID NO: 112 Of the clonesexamined above, 2 contained the expected sequence but with a single basemutation. Clone 54 (also designated pMF195) had an T base at position605 in SEQ ID NO: 112 in place of the A base, whereas clone 68 (alsodesignated pMF198) had a C base at position 1825 instead of the expectedT base. The sequence shown in SEQ ID NO: 112 was prepared from pMF195and and pMF198 by digesting both (10 μg of each) with XmaI (10 u) andXbaI (100 u) (New England Biolabs) in buffer (100 μl) containing 20 mMTris acetate (pH7.9) 50 mM potassium acetate. 10 mM Mg acetate. 1 mM DTTand BSA (100 μg/ml). The 215 base pairs fragment from pMF195 and thevector fragment from pMF198 (following treatment with alkalinephosphatase) were isolated from a 1% agarose gel and ligated together asdescribed previously. The ligation mix was used to transform competentDH5α. cells. The transformed cells were plated onto L agar plusampicillin and resulting colonies screened by digestion of the DNA withXmaI and XbaI for the presence of 5400 base pairs and 215 base pairsfragments. Positive clones were used for large scale plasmid DNApreparation and the sequence of the insert confirmed by DNA sequenceanalysis. The plasmid containing the 806.077 Fd-[A248S,G251T,D253K]HCPBgene from clone number 102 was named pMF213. The HindIII-XbaI fragmentfrom pMF213 was cloned into pEE6 [this is a derivative ofpEE6.hCMV—Stephens and Cockett (1989) Nucleic Acids Research 17, 7110—inwhich a HindIII site upstream of the hCMV promoter has been converted toa BglII site] in DH5α (screened by PCR with oligonucleotides SEQ ID NOS:106 and 107 for a 2228 base pairs insert) to give pMF221.

(b) Preparation of a Co-Expression Vector for Expression ofAntibody-Enzyme Fusion Protein

To generate vectors capable of expressing the antibody-enzyme fusionprotein in eukaryotic cells, the GS-System™ (Celltech Biologics) wasused (WO 87/04462, WO 89/01036, WO 86/05807 and WO 89/10404). Theprocedure requires cloning the humanised antibody light chain gene intothe HindIII-XmaI region of vector pEE14. This vector is described byBebbington in METHODS: A Companion to methods in Enzymology (1991) 2,136-145. To construct the expression vector, plasmids pEE14 andpNG3-VKss-806.077HuVK4-HuCK-Neo (Example 14) were digested with HinIIIand XmaI as described above. The appropriate vector (from pEE14) andinsert (732 base pairs from pNG3-VKss-806.077HuVK4-HuCK-Neo) from eachdigest were isolated from a 1% agarose gel and ligated together and usedto transform competent DH5α cells. The transformed cells were wereplated onto L agar plus ampicillin (100 μg/ml). Colonies were screenedby restriction analysis of isloated DNA for the presence of a 732 basepairs fragment on digestion of the DNA with HindIII and XmaI. Clonesproducing a 732 base pairs restriction fragment were used for largescale plasmid DNA preparation and the sequence of the insert confirmedby DNA sequence analysis. The plasmid containing the humanised lightchain sequence of SEQ ID NO: 70 in pEE14 was namedpEE14-806.077HuVK4-HuCK.

To make the co-expression vector, pMF221 (10 μg) was cut with BglII (20u) and SalI (40 U) in buffer (100 μl) containing 10 mM Tris-HCl (pH7.9). 150 mM NaCl, 10 mM MgCl₂, 1 mM DTT and BSA (100 μg/ml) and the4560 base pairs fragment isolated by agarose gel electrophoresis andpurified. Similarly, pEE14-806.077HuVK4-HuCK was cut with BamHI (40 u)and SalI (40 u) and the 9.95 kb vector fragment isolated and ligated tothe BglII-SalI fragment from pMF221 and cloned into DH5α. Colonies werescreened by PCR with 2 sets of oligonucleotides (SEQ ID NOS: 104 and105, and SEQ ID NOS: 108 and 109). Clones giving PCR products of 185base pairs and 525 base pairs respectively were characterised by DNAsequencing. A clone with the correct sequence was named pMF228—lightchain/Fd-mutant HCPB co-expression vector in DH5α. The humanisedFd-mutant HCPB sequence is shown in SEQ ID NO: 113. Residues 1 to 19 arethe signal sequence, residues 20 to 242 are the humanised variable andIgG3 CH1 region, residues 243 to 306 are the IgG3 hinge region andresidues 307 to 613 are the mutant HCPB sequence with the changes atresidues 248, 251 and 253 from the human HCPB sequence. The changes inthe HCPB sequence occur in SEQ ID NO: 113 at postions 554 (Ser), 557(Thr) and 559 (Lys) respectively.

(c) Preparation of a Vector for Expression of the Pro Domain of ProHCPB

A second eukaryotic expression plasmid, pEE12 containing a gene for theprepro sequence, for secretion of the pro domain with an additionalC-terminal leucine residue (termed pro-L), of preproHCPB was prepared asdescribed in Reference Example 17 of International Patent ApplicationNumber WO 96/20011. Plasmid pMF161 was prepared by PCR from pMF18 asdescribed for the unmodified prepro sequence, but using oligonucleotidesSEQ ID NOS: 110 and 111. The 359 base pairs fragment was cloned intopBluescript to give pMF141 and subsequently into pEE12 to give pMF161.The protein sequence of pro-L is shown in SEQ ID NO: 114.

(d) Expression of Antibody-Enzyme Fusion Protein in Eukaryotic Cells

For expression in eukaryotic cells, vectors containing genes capable ofexpressing the antibody enzyme-fusion protein (pMF228) and the pro-Lsequence (pMF161) were co-transfected into COS-7 cells. COS cells are anAfrican green monkey kidney cell line, CV-1, transformed with anorigin-defective SV40 virus and have been widely used for short-termtransient expression of a variety of proteins because of their capacityto replicate circular plasmids containing an SV40 origin of replicationto very high copy number. There are two widely available COS cellclones, COS-1 and COS-7. The basic methodology for transfection of COScells is described by Bebbington in Methods: A Companion to Methods inEnzymology (1991) 2, p. 141. For expression of HCPB, the plasmid vectorspMF48 and pMF67 (2 μg of each) were used to transfect the COS-7 cells(2×10⁵) in a six-well culture plate in 2 ml Dulbecco's Modified Eagle'sMedium (DMEM) containing 10% heat inactivated foetal calf serum (FCS) bya method known as lipofection—cationic lipid-mediated delivery ofpolynucleotides [Felgner et al. in Methods: A Companion to Methods inEnzymology (1993) 5, 67-75]. The cells were incubated at 37° C. in a CO₂incubator for 20 h. The mix of plasmid DNA in serum-free medium (200 μl)was mixed gently with LIPOFECTIN™ reagent (12 μl) and incubated atambient temperature for 15 min. The cells were washed with serum-freemedium (2 ml). Serum-free medium (600 μl) was added to theDNA/LIPOFECTIN™ and the mix overlaid onto the cells which were incubatedat 37° C. for 6 h in a CO₂ incubator. The DNA containing medium wasreplaced with normal DMEM containing 10% FCS and the cells incubated asbefore for 72 h. Cell supernatants (diluted 1:10 with 0.025M Tris-HClpH7.5; 125 μl) were analysed for activity against Hipp-Glu (5 h assay,in a total volume of 250 μl) essentially as described in Example 103.The diluted supernatant resulted in 18.4% hydrolysis of the Hipp-Glusubstrate.

Alternatively, the unmodified pro domain (from plasmid pMF67 describedin Reference Example 17 of International Patent Application Number WO96/20011) can be used in place of the pro-L expression plasmid in theabove experiment.

Large scale expression of proteins from COS cells is described by Ridderet al. (1995) in GENE 166, 273-276 and by Blasey et al. (1996) inCRYOTECHNOLOGY 18, 183-192.

For stable expression in CHO cells, the procedures described byBebbington in METHODS: A Companion to Methods in Enzymology (1991) 2,136-145 using GS selection with 25 μM and 50 μM MSX are followed.Alternatively, lipofection, essentially as described above fortransfection of COS cells may also be used to transfect CHO cells. Thecells are transfected with a mixture of plasmids pMF228 and pMF161 orpMF228 and pMF67. Supernatants from surviving colonies are screened byCEA ELISA (described in Example 11) and Western analysis (describedbelow) for the presence of a 70kDa band corresponding to the requiredantibody enzyme fusion protein. Supernatants, suitably diluted, are alsoscreened for enzyme activity as described in Example 103. Coloniesexpressing the desired antibody enzyme fusion protein are cultured atthe required scale (see for Example the publication by M E Reff (1993)in Current Opinion in Biotechnology 4, 573-576 and references citedtherein) and fusion protein purified from cell culture supernatant byone or more of the methods described in Example 102. (e) Westernanalysis Western blot analysis was performed as described as follows.Aliquots (20 μl) of each supernatant sample were mixed with an equalvolume of sample buffer (62.5 mM Tris, pH6.8, 1% SDS, 10% sucrose and0.05% bromophenol blue) with and without reductant. The samples wereincubated at 65° C. for 10 minutes before electrophoresis on a 8-18%acrylamide gradient gel (EXCEL™ gel system from Pharmacia BiotechnologyProducts) in a MULTIPHOR™ II apparatus (LKB Produkter AB) according tothe manufacturer's instructions. After electrophoresis, the separatedproteins were transfered to a membrane (HYBOND™ C-Super,AmershamInternational) using a NOVABLOU™ apparatus (LKB Produkter AB) accordingto protocols provided by the manufacturer. After blotting, the membranewas air dried.

The presence of antibody fragments was detected by the use of ananti-human kappa antibody (Sigma A7164, goat anti-human Kappa lightchain peroxidase conjugate) used at 1:2500 dilution. The presence ofhuman antibody fragments was visualised using a chemiluminescence system(ECL™ detection system, Amersham International).

EXAMPLES 49-74

Preparation of other Humanised 806.077 F(ab′)₂-Mutant HCPB FusionProteins

These Examples describe preparation of genes encoding humanised Fd heavychain fragments of 806.077 linked to a mutant HCPB (D253K; G251T,D253K;A248S,G25 1T,D253K) and their co-expression with a gene encoding ahumanised light chain of 806.077 and a gene encoding the pro domain ofhuman carboxypeptidase B to give the F(ab′)₂ protein with a molecule ofmutant HCPB at the C-terminus of each of the heavy chain fragments. Theconstant and hinge regions of of the humanised Fd heavy chain fragmentare derived from the human IgG1 or IgG2 or IgG3 antibody isotype. Theexpressed proteins are also referred to as antibody-enzyme fusionproteins.

The procedures described in Example 48 are repeated with the appropriatesequences derived from the table shown below. Oligonucleotides for PCRconstructions and clone screening are readily derived from theappropriate sequences.

To change the mutant HCPB sequence, the PCR template, plasmid pZEN1921,in part (a) of Example 48 is replaced with pZEN1860 for[G251T,D253K]HCPB (described in Reference Example 1) or pICI1713 for[D253K]HCPB (described in International Patent Application Number WO96/20011).

To change the antibody heavy chain constant and hinge region, the PCRtemplate, vector IgG3-pBSIIKS+, in part (a) of Example 48 is replacedwith pNG4-VHss-HulgG1CH1′ (described in Examples 39-47) orpNG4-VHss-HulgG2CH1′ (NCIMB No.40797).

To change the humanised antibody light chain sequence, the vectorpEE14-806.077HuVK4-HuCK in part (b) of Example 48 is replaced withpEE14-806.077HuVK1-HuCK or pEE14-806.077HuVK3-HuCK. The vectorspEE14-806.077HuVK1-HuCK and pEE14-806.077HuVK3-HuCK are prepared asdescribed for pEE14-806.077HuVK4-HuCK in part (b) of Example 48 butusing the 732 base pairs HindIII-XmaI fragment frompNG-VHss-806.077HuVK1-Neo and pNG-VHss-806.077HuVK3-Neo respectively(described in Examples 12-38) in place of the HindIII-XmaI fragment frompNG-VHss-806.077HuVK4-Neo.

Antibody-enzyme fusion protein variants for each Example are shown inthe table below.

TABLE Example Humanised Humanised Mutant HCPB No. Heavy chain Lightchain Enzyme 49 HuVH1-HuIgG3 HuVK4-HuCK [D253K]HCPB 50 HuVH1-HuIgG3HuVK4-HuCK [G251T,D253K]HCPB 51 HuVH1-HuIgG3 HuVK1-HuCK[A248S,G251T,D253K]HCPB 52 HuVH1-HuIgG3 HuVK1-HuCK [D253K]HCPB 53HuVH1-HuIgG3 HuVK1-HuCK [G251T,D253K]HCPB 54 HuVH1-HuIgG3 HuVK3-HuCK[A248S,G251T,D253K]HCPB 55 HuVH1-HuIgG3 HuVK3-HuCK [D253K]HCPB 56HuVH1-HuIgG3 HuVK3-HuCK [G251T1D253K]HCPB 57 HuVH1-HuIgG1 HuVK4-HuCK[A248S,G251T,D253K]HCPB 58 HuVH1-HuIgG1 HuVK4-HuCK [D253K]HCPB 59HuVH1-HuIgG1 HuVK4-HuCK [G251T,D253K]HCPB 60 HuVH1-HuIgG1 HuVK1-HuCK[A248S,G251T,D253K]HCPB 61 HuVH1-HuIgG1 HuVK1-HuCK [D253K]HCPB 62HuVH1-HuIgG1 HuVK1-HuCK [G251T,D253K]HCPB 63 HuVH1-HuIgG1 HuVK3-HuCK[A248S,G251T,D253K]HCPB 64 HuVH1-HuIgG1 HuVK3-HuCK [D253K]HCPB 65HuVH1-HuIgG1 HuVK3-HuCK [G251T,D253K]HCPB 66 HuVH1-HuIgG2 HuVK4-HuCK[A248S,G251T,D253K]HCPB 67 HuVH1-HuIgG2 HuVK4-HuCK [D253K]HCPB 68HuVH1-HuIgG2 HuVK4-HuCK [G251T,D253K]HCPB 69 HuVH1-HuIgG2 HuVK1-HuCK[A248S,G251T,D253K]HCPB 70 HuVH1-HuIgG2 HuVK1-HuCK [D253K]HCPB 71HuVH1-HuIgG2 HuVK1-HuCK [G251T,D253K]HCPB 72 HuVH1-HuIgG2 HuVK3-HuCK[A248S,G251T,D253]HCPB 73 HuVH1-HuIgG2 HuVK3-HuCK [D253K]HCPB 74HuVH1-HuIgG2 HuVK3-HuCK [G251T,D253K]HCPB

EXAMPLE 75

Preparation of [A248S,G251T,D253K]HCPB-(Humanised 806.077)F(ab′)₂ FusionProtein

This Example describes the preparation of a gene encoding pro-[A248S,G251T,D253K]HCPB linked to a humanised (version 1 VH with Human IgG3) Fdheavy fragment of antibody 806.077, and its co-expression with a geneencoding a humanised light chain (version 4 VK with CK) of the 806.077antibody. This gives the F(ab′)₂ protein with a molecule of thepro-[A248S,G25 1 T,D253K]HCPB at the N-terminus of each of the heavychain fragments. The enzyme is activated by the enzymatic removal of thepro domain using trypsin.

Standard molecular biology techniques, such as restriction enzymedigestion, ligation, kinase reactions, dephosphorylation, polymerasechain reaction (PCR), bacterial transformations, gel electrophoresis,buffer preparation and DNA generation, purification and isolation, werecarried out as described by Maniatis et al., (1989) Molecular Cloning, ALaboratory Manual; Second edition: Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., or following the recommended procedures ofmanufacturers of specific products. In most cases enzymes were purchasedfrom New England BioLabs, but other suppliers, and equivalent proceduresmay be used. Oligonucleotide sequences were prepared in an AppliedBiosystems 380A DNA synthesiser from 5′dimethoxytrityl base-protectednucleoside-2-cyanoethyl-N,N′-di-isopropyl-phosphoramidites and protectednucleoside linked to controlled-pore glass supports on a 0.2 μmol scale,according to the protocols supplied by Applied Biosystems Inc.

Mutants of HCPB, native HCPB and HCPB fusion proteins were assayed fortheir ability to convert hippuryl-L-glutamic acid or hippuryl-L-arginineacid to hippuric acid using an HPLC based assay as described in Example103 or International Patent Application Number WO 96/20011 Example 20.

Immunoassay techniques were carried out using methods based on thosedescribed by Tijssen, (1985) Practice and Theory of Enzyme Immunoassays,Laboratory Techniques in Biochemistry and Molecular Biology Volume 15,Elsevier Science Publishers, Amsterdam, or following the recommendedprocedures of manufacturers of specific products.

To generate plasmids capable of expressing the antibody-enzyme fusionprotein in eukaryotic cells the GS-System (Celltech Biologics) was used(details in International Patent Application Numbers WO 87/04462, WO89/01036, WO 86/05807 and WO 89/10404) with the two plasmids pEE6 (aderivative of pEE6.hCMV in which the HindIII restriction site upstreamof the hCMV promoter has been converted to a BglII site {Stephens andCockett, 1989, Nucleic Acids Research, 17, 71 10}) and pEE12 (aderivative of pSV2.GS with a number of restriction sites removed{Bebbington et al, 1992, Bio/Technology, 10. 169}).

a) Cloning Pre-Pro-HCPB up to Restriction Enzyme XmaI Cut Site (Position1048 in SEQ ID NO: 124)

Double stranded DNA of plasmid pMF18 (as described in InternationalPatent application Number WO 96/20011 Reference Example 19), a constructconsisting of pre-pro-HCPB cloned into vector pBluescript IIKS+(Stratagene), was prepared using standard DNA technology (Qiagenplasmid kit or similar), and restriction digested with HindIII and XmaIenzymes, being very careful to ensure complete digestion. Restrictionenzyme HindIII cuts the pMF18 plasmid just prior to the start of thepre-sequence of the HCPB gene, and XmaI cuts at the codon for amino acid240 (proline) of the mature protein, the HindIII to XmaI DNA piece isreferred to as the pre-pro-HCPB fragment. DNA of the correct size,containing the pre-pro-HCPB fragment (about 1061 base pairs) waspurified.

Double stranded DNA of plasmid vector pUC19 (New England BioLabs) wasprepared, restriction digested with HindIII and XmaI, and purified(about 2651 base pairs) in a similar manner to the pre-pro-HCPBfragment. Ligation mixes were prepared to clone the HCPB gene fragmentinto the pUC19 vector, using a molar ratio of about 1 vector to 2.5insert, and a final DNA concentration of about 2.5 ng/ml, in thepresence of T4 DNA ligase. 1 mM ATP and enzyme buffer. Following theligation reaction the DNA mixture was used to transform E.coli strainDH5α. Cell aliquots were plated on L-agar nutrient media containing 100μg/ml ampicillin as selection for plasmid vector, and incubatedovernight at 37° C. A number of colonies were picked and used formini-preparations of double stranded plasmid DNA. These DNA samples wereanalysed by restriction enzyme digestion, and a construct of the correctconfiguration identified. This plasmid containing the pre-pro-HCPBfragment up to the XmaI site in the mature gene is known as pCF003.

b) Cloning [A248S,G251T,D253K]HCPB from Position G241+Linker and 5 AminoAcids of VH

To separate the HCPB from the Fd sequence a neutral peptide linkerconsisting of (Glycine-Glycine-Glycine-Serine)₃ was introduced into thesequence during the PCR. In order to generate the fragment of the mutant[A248S,G251T,D253K]HCPB sequence (as documented in Reference Example 2)and add the peptide linker and the first 5 amino acids of the humanised806.077 VH, a PCR was set up using 100 pMols of primers CME 00971 andCME 00972 (SEQ ID NOs: 122 and 123) in the presence of approximately 5ng of pZen1921 DNA, dNTPs to a final concentration of 200 μM, Taqpolymerase reaction buffer, and 2.5 U of Taq polymerase in a finalvolume of 100 μl. The mixture was heated at 94° C. for 10 minutes priorto addition to the Taq enzyme, and the PCR incubation was carried outusing 30 cycles of 94° C. for 1.5 minutes, 55° C. for 2 minutes, and 72°C. for 2 minutes, followed by a single incubation of 72° C. for 10minutes at the end of the reaction. The PCR product containing the[A248S,G25 1 T,D253K]HCPB fragment (about 298 base pairs) was analysedfor DNA of the correct size by agarose gel electrophoresis and found tocontain predominantly a band of the correct size. The remainder of theproduct from the reaction mix was purified and separated from excessreagents using a microconcentrator column (Centricon™ 100, Amicon),followed by DNA isolation by ethanol/sodium acetate precipitation,centrifugation, vacuum drying and re-suspension in distilled water. Theisolated DNA was restriction digested with enzymes XmaI and EcoRI, and aband of the correct size (about 271 base pairs) purified.

Double stranded DNA of plasmid pCF003 (described above) prepared usingstandard DNA technology (Qiagen plasmid kits or similar), wasrestriction digested with XmaI and EcoRI enzymes, and a band of thecorrect size (about 2696 base pairs) purified.

Ligation mixes were prepared to clone the mutant HCPB gene fragment intothe vector, using a molar ratio of about 1 vector to 2.5 insert (1pCF003 to 2.5 [A248S,G251T,D253K]HCPB fragment PCR product), and a finalDNA concentration of about 2.5 ng/ml, in the presence of T4 DNA ligase,1 mM ATP and enzyme buffer. Following the ligation reaction the DNAmixture was used to transform E.coli strain DH5α. Cell aliquots wereplated on L-agar nutrient media containing 100 μg/ml ampicillin asselection for plasmid vector, and incubated overnight at 37° C. About200 colonies were picked and plated onto duplicate sterilenitro-cellulose filters (Schleicher and Schull), pre-wet on plates ofL-agar nutrient media containing 100 μg/ml ampicillin as selection forplasmid vector, and incubated overnight at 37° C. One duplicate platewas stored at 4° C., and acted as a source of live cells for thecolonies, the other plate was treated to denature and fix the DNA fromthe individual colonies to the nitro-cellulose. The nitro-cellulosefilter was removed from the agar plate and placed in succession ontofilter papers (Whatman) soaked in: 1.10% SDS for 2 minutes; 2. 0.5MNaOH, 1.5M NaCl for 7 minutes; 3. 0.5M NaOH, 1.5M NaCl for 4 minutes; 4.0.5M NaOH, 1.5M NaCl for 2 minutes; 5. 0.5M Tris pH7.4, 1.5M NaCl for 2minutes; and 6. 2×SSC (standard saline citrate) for 2 minutes. Thefilter was then placed on a filter paper (Whatman) soaked in 10×SSC andthe denatured DNA was crossed linked to the nitro-cellulose by ultraviolet light treatment (Spectrolinker XL-1500 UV crosslinker). Thefilters were allowed to air dry at room temperature, and were thenpre-hybridised at 60° C. for one hour in a solution of 6×SSC with gentleagitation (for example using a Techne HB-1D hybridizer). Notepre-hybridisation blocks non-specific DNA binding sites on the filters.

In order to determine which colonies contain DNA inserts of interest theDNA cross-linked to the nitro-cellulose filter was hybridised with aradio-labelled ³²P-DNA probe prepared from the [A248S,G251T,D253K]HCPBpurified PCR DNA fragment (see above). About 50 ng of DNA was labelledwith 50 μCi of ³²P-dCTP (>300 μCi/mMol) using T7 DNA polymerase in atotal volume of 50 μl (Pharmacia T7 Quickprime kit), and the reactionallowed to proceed for 15 minutes at 37° C. The labelled probe washeated to 95° C. for 2 minutes, to denature the double stranded DNA,immediately added to 10 ml of 6×SSC at 60° C., and this solution wasused to replace the pre-hybridisation solution on the filters.Incubation with gentle agitation was continued for about 3 hours at 60°C. After this time the hybridisation solution was drained off, and thefilters were washed twice at 60° C. in 2×SSC for 15 minutes each time.Filters were then gently blotted dry, covered with cling film (Saran™wrap or similar), and exposed against X-ray film (for example KodakX-OMAT-ARS™) overnight at room temperature. Following development of thefilm, colonies containing inserts of interest were identified as thosewhich gave the strongest exposure (darkest spots) on the X-ray film. Inthis series of experiments about 15% of the colonies gave positivehybridisation. From this 12 colonies were chosen for further screening.These colonies were picked from the duplicate filter, streaked andmaintained on L-agar nutrient media containing 100 μg/ml ampicillin, andgrown in L-broth nutrient media containing 100 μg/ml ampicillin.

The selected colonies were used for mini-preparations of double strandedplasmid DNA. These DNA samples were analysed by restriction enzymedigestion, and constructs of the correct configuration identified. Inorder to ensure that no changes to the DNA sequence had been introducedduring the PCR a number of clones with correct restriction mapping weretaken for DNA preparation using standard technology (Qiagen plasmid kitsor similar), and the inserts sequenced using several separateoligonucleotide primers. A construct of the correct sequence wasidentified, and this plasmid containing thepre-pro-[A248S,G251T,D253K]HCPB-linker-humanised 806.077 VH gene up tothe PstI site (at amino acid 5)(position 1301 in SEQ ID NO: 124) istermed pCF004.

c. Cloning Humanised 806.077 Fd

Double stranded DNA of plasmid pNG4-VHss-HuVH1-806.077-IgG3CH1′, aconstruct consisting of the humanised 806.077 version 1 VH with humanIgG3 CH1 and hinge region cloned into vector pNG4 (see Example 44), wasprepared using standard DNA technology (Qiagen plasmid kit or similar),and restriction digested with PstI and XmaI enzymes. DNA of the correctsize, containing the humanised 806.077 Fd fragment (about 854 basepairs) was purified. Double stranded DNA of plasmid vector pUC19 (NewEngland BioLabs) was prepared, restriction digested with PstI and XmaI,and purified (about 2659 base pairs) in a similar manner to thehumanised 806.077 Fd fragment.

Aliquots of both restricted and purified DNA samples were checked forpurity and concentration estimation using agarose gel electrophoresiscompared with known standards. From these estimates ligation mixes wereprepared to clone the humanised 806.077 Fd gene fragment into the pUC19vector, using a molar ratio of about 1 vector to 2.5 insert, and a finalDNA concentration of about 2.5 ng/ml, in the presence of T4 DNA ligase,1 mM ATP and enzyme buffer.

Following the ligation reaction the DNA mixture was used to transformE.coli strain DH5α. Cell aliquots were plated on L-agar nutrient mediacontaining 100 μg/ml ampicillin as selection for plasmid vector, andincubated overnight at 37° C. A number of colonies were picked and usedfor mini-preparations of double stranded plasmid DNA. These DNA sampleswere analysed by restriction enzyme digestion, and a construct of thecorrect configuration identified. This plasmid containing the humanised806.077 Fd fragment from the PstI site to the XmaI site is known aspCF005.

d) Cloning Humanised 806.077 Fd intoPre-Pro-[A248S,G251T,D253K]HCPB-Linker Construct

Double stranded DNA of plasmid pCF005 (as documented above), wasprepared using standard DNA technology (Qiagen plasmid kit or similar),and restriction digested with PstI and EcoRI enzymes. DNA of the correctsize, containing the humanised 806.077 Fd fragment (about 870 basepairs) was purified. Double stranded DNA of plasmid vector pCF004 (asdocumented above) was prepared, restriction digested with PstI andEcoRI, and purified (about 3950 base pairs) in a similar manner to thehumanised 806.077 Fd fragment. Ligation mixes were prepared to clone thehumanised 806.077 Fd gene fragment into the pCF004 vector, using a molarratio of about 1 vector to 2.5 insert, and a final DNA concentration ofabout 2.5 ng/μl, in the presence of T4 DNA ligase, 1 mM ATP and enzymebuffer.

Following the ligation reaction, the DNA mixture was used to transformE.coli strain DH5α. Cell aliquots were plated on L-agar nutrient mediacontaining 100 μg/ml ampicillin as selection for plasmid vector, andincubated overnight at 37° C. A number of colonies were picked and usedfor mini-preparations of double stranded plasmid DNA. These DNA sampleswere analysed by restriction enzyme digestion, and a construct of thecorrect configuration identified. This plasmid containing thepre-pro-[A248S,G251T,D253K]HCPB-Linker-Fd(humanised 806.077) in pUC19 isknown as pCF006.

e) Cloning Pre-Pro-[A248S,G251 T,D253K]HCPB-Linker-(Humanised 806.077)Fdinto pEE6 hCMV Vector

Double stranded DNA of plasmid pCF006 (as documented above), wasprepared using standard DNA technology (Qiagen plasmid kit or similar),and restriction digested with HindIII and EcoRI enzymes. DNA of thecorrect size, containing the fusion protein (about 2185 base pairs) waspurified.

Double stranded DNA of plasmid vector pEE6 (as documented above) wasprepared, restriction digested with HindIII and EcoRI, and purified(about 4775 base pairs) in a similar manner to the fusion protein.Ligation mixes were prepared to clone the humanised 806.077 Fd fusionprotein into the pEE6 vector, using a molar ratio of about 1 vector to2.5 insert, and a final DNA concentration of about 2.5 ng/μl, in thepresence of T4 DNA ligase, 1 mM ATP and enzyme buffer. Following theligation reaction the DNA mixture was used to transform E.coli strainDH5α. Cell aliquots were plated on L-agar nutrient media containing 100μg/ml ampicillin as selection for plasmid vector, and incubatedovernight at 37° C. A number of colonies were picked and used formini-preparations of double stranded plasmid DNA. These DNA samples wereanalysed by restriction enzyme digestion, and a construct of the correctconfiguration identified. This plasmid containing thepre-pro-[A248S,G251T,D253K]HCPB-Linker-Fd(humanised 806.077) in pEE6 isknown as pCF007.

f) Cloning Humanised 806.077 Light Chain Version 4 into pEE12 Vector

Double stranded DNA of plasmid pNG3-VKss-806.077-HuVK4-HuCK-Neo. aconstruct consisting of the humanised 806.077 version HuVK4 with humanCK cloned into vector pNG3 (see Examples 12-38), was prepared usingstandard DNA technology (Qiagen plasmid kit or similar), and restrictiondigested with HindIII and EcoRI enzymes. DNA of the correct size,containing the humanised 806.077 light chain (about 2022 base pairs) waspurified. Double stranded DNA of plasmid vector pEE12 was prepared,restriction digested with HindIII and EcoRI, and purified (about 7085base pairs) in a similar manner to the humanised 806.077 light chain.Ligation mixes were prepared to clone the humanised 806.077 light chaininto the pEE12 vector, using a molar ratio of about 1 vector to 2.5insert, and a final DNA concentration of about 2.5 ng/μl, in thepresence of T4 DNA ligase, 1 mM ATP and enzyme buffer. Following theligation reaction the DNA mixture was used to transform E.coli strainDH5α. Cell aliquots were plated on L-agar nutrient media containing 100μg/ml ampicillin as selection for plasmid vector, and incubatedovernight at 37° C. A number of colonies were picked and used formini-preparations of double stranded plasmid DNA. These DNA samples wereanalysed by restriction enzyme digestion, and a construct of the correctconfiguration identified. This plasmid containing the humanised 806.077light chain version 4 is known as pCF008/4.

g) Cloning CMVp-Pre-Pro-[A248S, G251 T, D253K]HCPB-Linker-Humanised 806.077)Fd into pCF008/4.

Double stranded DNA of plasmid pCF007 (as documented above), wasprepared using standard DNA technology (Qiagen plasmid kit or similar),and restriction digested with BglII and SalI enzymes. Restriction enzymeBglII cuts the pCF007 plasmid prior to the start of the CMV MIE leader,promoter and gene for the fusion protein. Restriction enzyme SalI cutsabout 520 base pairs after the stop codons of the mature protein. DNA ofthe correct size, containing the fusion protein (about 4844 base pairs)was purified. Double stranded DNA of plasmid vector pCF008/4 wasprepared, restriction digested with BamHI and SalI, and purified (about7436 base pairs) in a similar manner to the fusion protein. Ligationmixes were prepared to clone the[A248S,G251T,D253K]HCPB-linker-(humanised 806.077)Fd fusion gene intothe pCF008/4 vector, using a molar ratio of about 1 vector to 2.5insert, and a final DNA concentration of about 2.5 ng/μl, in thepresence of T4 DNA ligase, 1 mM ATP and enzyme buffer. Following theligation reaction the DNA mixture was used to transform E.coli strainDH5α. Cell aliquots were plated on L-agar nutrient media containing 100μg/ml ampicillin as selection for plasmid vector, and incubatedovernight at 37° C. A number of colonies were picked and used formini-preparations of double stranded plasmid DNA. These DNA samples wereanalysed by restriction enzyme digestion, and a construct of the correctconfiguration identified. This plasmid containing genes forpro-[A248S,G251T,D253K]HCPB-Linker-F(ab′)₂(humanised 806.077 antibody)in the GS expression vector pEE12 is known as pCF009 and a plasmid mapis shown in FIG. 2. The DNA and amino acid sequences of the light chainHuVK4 are shown in SEQ ID NOs: 70 and 71. The DNA sequence of thepre-pro-[A248S,G251T,D253K]HCPB-linker-Fd(Humanised 806.077) is shown inSEQ ID NO: 124 and the corresponding amino acid sequence in SEQ ID NO:125.

h) Expression of Pro-[Mutant]HCPB-Linker-F(ab′)₂(Humanised 806.077) fromMouse Myeloma Cells.

The following method has been used for myeloma expression of all (D253Kand G251T,D253K and A248S,G251T,D253K) mutant pro-HCPB enzyme fusionproteins. The preferred mouse myeloma cell line is NS0 (Galfre andMilstein, 1981, Methods in Enzymol., 73, 346), and is available form theEuropean Collection of Animal Cell Cultures, PHLS CAMR, Porton Down,Salisbury, Wiltshire, SP4 0JG (ECACC catalogue number 85110503). Thesecells were grown in Dulbecco's Modified Eagle Medium (DMEM; Gibco/BRL)containing 10% heat inactivated foetal calf serum (FCS).

For expression of pro-[A248S,G251T,D253K]HCPB-linker-F(ab′)₂(humanised806.077) two plasmids were used, pCF009 (described above) and pRc/RSV(from Invitrogen, Cat no. V780-20) which contains the neomycinresistance gene for selection of G418 resistant stable cell lines. About5 μg of each plasmid (from 0.5 to 10 μg) were used to transfectapproximately 8×10⁶ NS0 cells by the method of lipofection (Felgner etal., in Methods: A Companion to Methods in Enzymology, 1993, 5, 67-75)which involves the cationic lipid mediated delivery of polynucleotidesinto eukaryotic cells. The cells were harvested by centrifugation,washed with serum free medium (30 ml), resuspended in 800 μl of mediumand kept at 37° C. in a tissue culture flask until the DNA was added.Serum-free medium (450 μl) was mixed gently with LIPOFECTIN™ reagent(5041) and incubated at room temperature for 30 to 45 minutes. Thismixture was added to 500 μl of medium containing the plasmid DNA mixture(in less than 100 μl) and left at room temperature for 15 minutes. Serumfree medium (600 μl) was added to the plasmid DNA-LIPOFECTIN™ mixture,and the complex added to the cells which were incubated for about 5hours at 37° C. in a CO₂ incubator. The DNA containing medium was thenreplaced with normal DMEM medium (8 ml) containing 10% FCS and the cellsincubated overnight. The medium was then again replaced with normal DMEMmedium (8 ml) containing 10% FCS and the cells incubated as previouslywithout selection for 24 hours. At the end of this period the medium waschanged to DMEM containing 10% FCS and G418 selection (1.5 mg/ml), andthe cells diluted (between 1 in 4 and 1 in 20) (approximately 0.5 to1.5×10⁶ cells per plate) in the same medium into micro-titre wells (150μl per well; 2 or more plates per dilution). The micro-titre plates wereincubated for at least two weeks at 37° C. in a CO₂ incubator and thenchecked regularly for viable clone formation.

Media from wells containing single viable clones was taken for testingand replaced with fresh media (containing G418). The removed media wastested for antibody binding to CEA in an ELISA (in the same manner asdescribed in International Patent application Number WO 96/2001 1Reference Example 5 part 1, except that the secondary antibody solutionwas changed from anti-mouse to anti-human (goat anti-human Kappa lightchain peroxidase conjugate, Sigma A7164). Positive samples for the CEAELISA were also tested for [A248S,G251T,D253K]HCPB enzyme activity (asdescribed above) following activation (removal of the pro domain fromthe fusion protein) by trypsin (700 μg/ml in 50 mM Tris-HCl and 150 mMNaCl pH 7.6 at 4° C. for 1 hour, the reaction being stopped by theaddition of a five fold excess of soy bean trypsin inhibitor). A numberof clones were identified which produced media that was positive forboth 806.077 antibody binding to CEA and [A248S,G251T,D253K]HCPB enzymeactivity. These were further tested by non-reducing Western blotanalysis (in the same manner as described in International PatentApplication Number WO 96/20011 Reference Example 5 part j, except thatthe antibody solution is changed from anti-mouse to anti-human (goatanti-human Kappa light chain peroxidase conjugate, Sigma A7164) toidentify clones which produce predominately F(ab′)₂(806.077) fusionprotein. These clones were then expanded, tested for stable generationof the fusion protein over a number of generations, and the highestproducers bulked up and stored frozen in liquid nitrogen using standardtechnology.

Amplification, high-level expression and fermentation of fusion proteinsfrom NSO myeloma cells was performed in a similar manner to thatdescribed by Bebbington et al. (1992) in Bio/Technology 10, 169-175.Fusion protein was purified, and the pro-sequence removed as describedin Example 102.

EXAMPLES 76 TO 101

Cloning and Expression of Other Variants of Pro-HCPB-Linker-(Humanised806.077)Fd+(Humanised 806.077) Light Chain

The method for the generation of fusion proteins with other mutants ofHPCB was similar to that detailed in Example 75 (above), with theexception that in part b. of Example 75 there was a substitution of[D253K]HCPB or [G251T,D253K]HCPB for [A248S,G251T,D253K]HCPB and theplasmid DNA used in the PCR reaction was pICI1713 (as described inInternational Patent application Number WO 96/20011, Example 15) orpZEN1860 (Reference Example l) respectively. After cloning,identification, and sequence confirmation the resulting plasmidcontaining pre-pro-[D253K]HCPB-linker orpre-pro-[G251T,D253K]HCPB-linker and humanised 806.077 VH gene up to thePstI site (at amino acid 5) in the pUC19 vector back ground was used inplace of pCF004 in the subsequent cloning reactions.

The method for generation of fusion proteins with other CH1 domains wassimilar to that detailed in Example 75 (above), with the exception thatin part c. of Example 75 there was a substitution of plasmids containingeither humanised 806.077 VH version 1 with human IgG1 or IgG2 CH1 andhinge regions in place of 806.077-HuVH1-IgG3CH1′ (SEQ ID NOs: 96 and 56respectively). After cloning, identification, and sequence confirmationthe resulting plasmid containing the IgG1 or IgG2 sequence was used inplace of pCF005 in the subsequent cloning reactions.

The method for generation of fusion proteins with other variants of thehumanised 806.077 light chain was similar to that detailed in Example 75(above), with the exception that in part f. of Example 75 there was asubstitution of plasmids containing either humanised 806.077 Lc version1 or version 3 in place of 806.077-HuVK4-HuCK (SEQ ID NOs: 51 and 96respectively). After cloning, identification, and sequence confirmationthe resulting plasmid containing the alternative light chain sequencewas used in place of pCF008/4 in the subsequent cloning reactions. Thefusion protein variants for each Example (76 to 101) are shown in thefollowing table.

TABLE Example Humanised Humanised Mutant HCPB No. Heavy chain Lightchain Enzyme 76 HuVH1-HuIgG3 HuVK4-HuCK [D253K]HCPB 77 HuVH1-HuIgG3HuVK4-HuCK [G25IT,D253K]HCPB 78 HuVH1-HuIgG3 HuVK1-HuCK[A248S,G251T,D253K]HCPB 79 HuVH1-HuIgG3 HuVK1-HuCK [D253K]HCPB 80HuVH1-HuIgG3 HuVK1-HuCK [G251T,D253K]HCPB 81 HuVH1-HuIgG3 HuVK3-HuCK[A248S,G251T,D253K]HCPB 82 HuVH1-HuIgG3 HuVK3-HuCK [D253K]HCPB 83HuVH1-HuIgG3 HuVK3-HuCK [G251T,D253K]HCPB 84 HuVH1-HuIgG1 HuVK4-HuCK[A248S,G251T,D253K]HCPB 85 HuVH1-HuIgG1 HuVK4-HuCK [D253K]HCPB 86HuVH1-HuIgG1 HuVK4-HuCK [G251T,D253K]HCPB 87 HuVH1-HuIgG1 HuVK1-HuCK[A248S,G251T,D253K]HCPB 88 HuVH1-HuIgG1 HuVK1-HuCK [D253K]HCPB 89HuVH1-HuIgG1 HuVK1-HuCK [G25IT,D253K]HCPB 90 HuVH1-HuIgG1 HuVK3-HuCK[A248S,G251T,D253K]HCPB 91 HuVH1-HuIgG1 HuVK3-HuCK [D253K]HCPB 92HuVH1-HuIgG1 HuVK3-HuCK [G251T,D253K]HCPB 93 HuVH1-HuIgG2 HuVK4-HuCK[A248S,G251T,D253K]HGPB 94 HuVH1-HuIgG2 HuVK4-HuCK [D253K]HCPB 95HuVH1-HuIgG2 HuVK4-HuCK [G251T,D253K]HCPB 96 HuVH1-HuIgG2 HuVK1-HuCK[A248S,G251T,D253K]HCPB 97 HuVH1-HuIgG2 HuVK1-HuCK [D253K]HCPB 98HuVH1-HuIgG2 HuVK1-HuCK [G25IT,D253K]HCPB 99 HuVH1-HuIgG2 HuVK3-HuCK[A248S,G251T,D253K]HCPB 100  HuVH1-HuIgG2 HuVK3-HuCK [D253K]HCPB 101 HuVH1-HuIgG2 HuVK3-HuCK [G251T,D253K]HCPB

EXAMPLE 102

Purification of Proteins Containing 806.077 Antibody Sequences

Purification or enrichment of recombinant F(ab′)₂ or antibody-enzymefusion proteins may be achieved from myeloma cell, CHO cell or COS cellsupernatants by several methods, used either singly or together.Purification of murine 806.077 F(ab′)₂, chimeric 806.077 F(ab′)₂constructs and fully humanised 806.077 F(ab′)₂ constructs, andantibody-enzyme fusion protein constructs incorporating these F(ab′)₂constructs were achieved by one or more of several different methods,affinity chromatography or anion exchange chromatography, or proteinA/protein G chromatography. These techniques can also be applied topurification of 806.077 antibody—B7 fusions (see Example 104).

a) Antigen Affinity Chromatography

Carcinoembryonic antigen (CEA), to which the parent murine 806.077antibody was raised, was immobilised on a column (using Pharmaciaproducts). In brief, immobilisation was via a stable ester bond toSepharose™ High Performance medium, NHS-activated prepacked in columns(HiTrap™); coupling of the CEA to the activated matrix was performedfollowing the standard instructions provided with the product.

Preparation of a 1 ml Affinity Column.

CEA stock solution (8 mg/ml) was first diluted with coupling buffer(0.2M sodium hydrogen carbonate, 0.5M sodium chloride; pH8.3) to a finalconcentration of 0.5 mg/ml. A new column was washed with 6 ml ofice-cold 1 mM HCl at a flow rate not exceeding 1 ml/min. Immediatelyafter, the CEA ligand (1 ml at 0.5 mg/ml) was injected onto the column.The column was sealed at both ends and left to stand for 30 minutes atroom temperature. Excess active groups that had not coupled to theligand were deactivated and any non-specifically bound ligand was washedout of the column by three rounds of alternating high and low pH washes.The buffers used were 0.5M ethanolamine, 0.5M sodium chloride (pH8.3)and 0.1M sodium acetate, 0.5M sodium chloride (pH 4.0). In each round ofwashes 6 ml of each buffer was washed over the column matrix. Finally,the column was washed into storage buffer (0.05M Na₂HPO₄, 0.1% NaN₃,pH7.0).

Purification Procedure

The cell culture supernatant containing the desired F(ab′)₂ or fusionconstruct e.g. chimeric 806.077 F(ab′)₂, humanised 806.077 F(ab′)₂, orantibody-enzyme fusion protein was diluted 1:1 with phosphate bufferedsaline (pH 7.2) and passed over the 1 ml affinity column at a flow rateof 1 m/min. The column had previously been equilibrated with phosphatebuffered saline (pH7.2; 50 mM sodium phoshate, 150 mM sodium chloride).The column was washed with 10 column volumes of phosphate bufferedsaline after the cell supernatant had passed over it. Bound F(ab′)₂ waseluted with 5 column volumes of 100 mM sodium citrate (pH3.0), with 1 mlfractions of the eluant being collected. Detection of the eluted F(ab′)₂was achieved by Western blot analysis using a suitable antibodyperoxidase conjugate (an anti-human Kappa Light chain-peroxidaseconjugate in the case of the fully humanised F(ab′)₂, Sigma A-7164) anddeveloping with hydrogen peroxide and 4-chloro-1-naphthol. Appropriatefractions were pooled and concentrated, using a centrifugal concentrator(Centricon™ 30), where necessary.

b) Anion Exchange Chromatography

Cell culture supernatant containing the required F(ab′)₂ or fusionconstruct e.g. chimeric 806.077 F(ab′)₂, humanised 806.077 F(ab′)₂, orantibody-enzyme fusion protein was diafiltered into 50 mM Tris (using astirred cell with a 10,000 molecular weight cut-off membrane) until theionic strength of the solution was equivilant to the columnequilibration buffer. The 40 ml aliquot of the diafiltered supernatantwas loaded on to a suitable column (Pharmacia Mono Q™ 10/10 HR) at 2ml/min. The column was previously equilibrated with 50 mM Tris (pH8.0).Once the supernatant had passed over the column, the column was washedback to baseline with the equilibration buffer. Bound material on thecolumn was then eluted with a 0-50% buffer B (50 mM Tris, 1M sodiumchloride pH8.0 ) over 15 column volumes. Elution fractions werecollected (4 ml per fraction) and those containing the F(ab′)₂ wereidentified by Western blot analysis using a suitable antibody peroxidaseconjugate (an anti-human Kappa Light chain -peroxidase conjugate in thecase of the fully humanised F(ab′)₂, Sigma A-7164) and developing withhydrogen peroxide and 4-chloro-1-naphthol. Appropriate fractions werepooled and concentrated using a centrifugal concentrator (Centricon™30), where necessary.

c) Protein A and Protein G Purification

The cell culture supernatant containing the desired F(ab′)₂ or fusionconstruct (e.g. 806.077 F(ab′)₂, chimeric 806.077 IgG₁ or IgG₂ or IgG₃;pro-HCPB-linker-806.077 F(ab′)₂ ,806.077 F(ab′)₂-HCPB) was diluted 1:1with phosphate buffered saline before being loaded on to a columnpreviously equilibrated in phosphate buffered saline (pH7.2). The columnwas washed with phosphate buffered saline, back to baseline, before thebound F(ab′)₂ or fusion protein was eluted with 100 mM sodium citrate(pH 3.0) in the case of the F(ab′)₂ and 50 mM glycine, 100 mM sodiumchloride (pH10.8) in the case of the fusion proteins. Elution fractionswere collected and neutralised by the addition of 125 μl 2M Tris per 1ml of elution volume. Those fractions containing the F(ab′)₂ were pooledand concentrated where necessary using a centrifugal concentrator.

d) Pro-Sequence Cleavage.

For fusion proteins containing a covalently linked pro-sequencee.g.(Pro-HCPB-linker-806.077 F(ab′)₂) the pro sequence was cleaved byincubation the fusion with trypsin. This procedure at a milligram (offusion) scale involved the following. Trypsin was mixed with the fusionprotein in a ratio of 1:1000 (trypsin:fusion). The mixture was incubatedfor 24 hours at room temperature (around 22° C.), after which thecleavage of the pro sequence was complete. The fusion protein wasseparated from the pro sequence by recirculating the mixture in one ofthe generic chromatography purification or enrichment protocols.

EXAMPLE 103

Assay of Activity of Antibody-Enzyme Fusion Proteins Containing MutantHuman CPB Against Hipp-Glu Prodrug Analogues

Cell culture supernatants or purified antibody-enzyme fusion proteinscontaining mutants of human CPB (D253K; G252T,D253K; A248S,G251T,D253K:Examples 48-101) are assayed for their ability to converthippuryl-L-glutamic acid (Hipp-Glu; Reference Example 9 in InternationalPatent Application Number WO 96/20011)) to hippuric acid using a HPLCbased assay.

The reaction mixture (250 μl) contains either 4 μg of purified fusionprotein or cell culture supernatant (used either neat or diluted with0.025M Tris-HCl pH7.5; 125 μl) and 0.5 mM Hipp-Glu in 0.025 M Tris-HCL,pH 7.5. Samples are incubated for 5 hr at 37° C. The reactions areterminated by the addition of 250 μl of 30% methanol, 70% phosphatebuffer (50 mM; pH 6.5), 0.2% trifluoroacetic acid and the amount ofhippuric acid generated is quantified by HPLC (using a Hewlett Packard1090 Series 11 with diode array system).

Samples (50 μl) are injected onto a column (25 cm; HICHROM™ Hi-RPB) andseparated using a mobile phase of 15% methanol, 85% phosphate buffer (50mM; pH 6.5) at a flow rate of 1 ml/min. The amount of product (hippuricacid) produced is determined from calibration curves generated withknown amounts of hippuric acid (Sigma-H6375). Results are expressed asthe percentage conversion of substrate into product at 37° C. at timesranging from 30 min-24 h depending on rate of conversion.

For antibody-enzyme fusion proteins with an N-terminal proCPB, the prodomain is first removed by treatment with trypsin (700 μg/ml) in 50 mMTris-HCl (pH7.6), 150 mM NaCl at 4° C. for 1 h.

EXAMPLE 104

Preparation of a Human B7.1-Humanised 806.077 F(ab′)₂ Fusion Protein(hB7-806)

As in Reference Example 3, a fusion protein consisting of the signalsequence and extracellular domain of human B7.1 fused directly to the 5′coding region of the humanised 806.077 antibody Fd chain is constructedusing PCR techniques. A HindIII-NheI fragment is created containing thenatural signal sequence and extracellular domain of human B7.1 fused tothe VH region of a humanised 806.077 antibody heavy chain. This iscloned into a suitable vector, for example pNG4-V_(H)ss-HuIgG2CH1′ orpNG4-V_(H)ss-HuIgG3CH1′ (see Examples 39-47) (replacing bases 1423 inSeq.ID NO: 18), to create a human B7.1-humanised 806.077 Fd fusion gene.Co-expression of this fusion with a humanised 806.077 L chain (asuitable vector containing the VK4 version of humanised 806.077 lightchain is pCF008/4; see Example 75) is then achieved after constructionof a co-expression vector using expression systems such as thosedescribed herein. Such a vector is used to transfect NSO myeloma cellsand colonies selected on the presence of CEA binding activity in theculture supernatant. Other humanised sequences are described in Examples39-47.

The hB7-806 fusion protein is expressed from a suitable cell line andpurified using protein-A column as described in Reference Example 3 orone of the methods described in Example 102. It should be noted thatpurification methods other than protein-A columns are preferred forhumanised 806.077 antibody fragments and fusion proteins thereof. Thefusion protein can be tested for both antigen and receptor bindingproperties and T-cell co-stimulatory activity when bound to LS174T cellsusing assays set out in Reference Example 3.

EXAMPLE 105

Preparation of Chimeric and Humanised 806.077 F(ab′)₂-CPG2 Conjugates

The procedure described in Example 5 was repeated with the murineF(ab′)₂ protein replaced by one of the chimeric versions described inExample 8 or one of the humanised versions described in Examples 39-47.

EXAMPLE 106

Preparation of Humanised 806.077 Fab-CPG2 Enzyme Fusion Protein.

Humanised 806.077 antibody and bacterial CPG2 enzyme fusion proteinconstructs are constructed using PCR methodology similar to thatdescribed for the construction of HuVK4 in Examples 12-38, in whichspecifically designed primers are used in a PCR reaction to amplify theantibody and enzyme gene components (such that the resulting DNAproducts contain overlapping complementary sequence) which are thenjoined via a further “splicing/joining” PCR reaction to make thecomplete antibody-enzyme fusion gene. The fusion protein is created byjoining the 3′ end of Fd humanised 806.077 antibody heavy chain gene tothe 5′ end of the CPG2 structural coding gene to create a Fab-CPG2fusion protein coding gene. In such a construct, the humanised 806.077antibody heavy chain gene component may be terminated after residue K236for the HuVH1-HuIgG1 Fd heavy chain (SEQ ID NO: 93), after residue Val237 for the HuVH1-HuIgG2 Fd heavy chain (SEQ ID NO: 57) or after residueVal 237 heavy chain in the HuVH1-HuIgG3 Fd heavy chain (SEQ ID NO: 95)(thus, in each case, excluding any sequence pertaining to the hingeregion) and may be joined to the first CPG2 residue positionedC-terminal to the signal sequence cleavage site (Minton et al (1984)Gene 31, 31-38). However, in order to obtain optimal antibody bindingand enzymatic properties, it is also envisaged that it may be desirableto incorporate additional residues at the junction between the twoconstituent components.

The fusion gene is then cloned into a suitable vector, for examplepNG4-VHss-HuIgG2CH1′ (NCIMB no. 40797), after the appropriate restictionenzyme digestion, isolation of the vector and fusion gene DNA fragmenthave been made thus replacing the original antibody gene with that ofthe fusion protein. Co-expression of the fusion with a humanised 806.077light chain is then achieved after construction of a co-expressionvector in a manner analogous to that described in Example 11. Theco-expression vector is used to transfect NSO myeloma cells and coloniesselected on the presence of CEA and Fd binding activity in the culturesupernatant as previously described. The fusion protein can be purifiedusing a Protein-A column and shown to have both antigen and enzymaticproperties using standard test methodology.

EXAMPLE 107

Further Combination of Humanised Heavy and Light Chain Variable RegionsBased on Light Chain Sequence VK4

The procedures described in Examples 12-38 are repeated with thehumanised light chain variable sequence of VK4 (SEQ ID NO: 71) replacedby the modified sequence in which the tyrosine residue (Tyr) at position35 of SEQ ID NO: 71 is replaced by a phenylalanine residue.(Phe).

EXAMPLE 108

Further Combination of Humanised Heavy and Light Chain Variable RegionsBased on Light Chain Sequence VK4

The procedures described in Examples 12-38 are repeated with thehumanised light chain variable sequence of VK4 (SEQ ID NO: 71) replacedby the modified sequence in which the phenylalanine residue (Phe atposition 72 of SEQ ID NO: 71 is replaced by a leucine residue (Leu).

EXAMPLE 109

Further Combination of Humanised Heavy and Light Chain Variable RegionsBased on Light Chain Sequence VK4

The procedures described in Examples 12-38 are repeated with thehumanised light chain variable sequence of VK4 (SEQ ID NO: 71) replacedby the modified sequence in which the tyrosine residue (Tyr) at position35 and the phenylalanine residue (Phe) at position 72 of SEQ ID NO: 71are replaced by a phenylalanine residue (Phe) and a leucine residue(Leu) respectively.

EXAMPLE 110

Combination of Humanised Heavy Chain Variable Regions and a ChimericLight Chain Sequence

The procedures described in Examples 12-38 are repeated with thehumanised light chain variable sequence of replaced by the chimericsequence of SEQ ID NO: 17 described in Example 8.

EXAMPLE 111-113

Expression of Humanised F(ab′)2 Fragments with a Modified Light ChainVK4 Variable Sequence

The procedures described in Examples 39-47 are repeated with thevariable light chain sequence described in Example 107 used to make areplacement for the humanised light chain sequence of SEQ ID NO: 99 inwhich the tyrosine residue (Tyr) at position 57 of SEQ ID NO: 99 isreplaced by a phenylalanine residue (Phe).

Example 111 is the combination of HuVH1-HuIgG1 and the modified SEQ IDNO: 99 described above.

Example 112 is the combination of HuVH1-HuIgG2 and the modified SEQ IDNO: 99 described above.

Example 113 is the combination of HuVH1-HuIgG3 and the modified SEQ IDNO: 99 described above.

EXAMPLE 114-116

Expression of Humanised F(ab′)₂ Fragments with a Modified Light ChainVK4 Variable Sequence

The procedures described in Examples 39-47 are repeated with thevariable light chain sequence described in Example 108 used to make areplacement for the humanised light chain sequence of SEQ ID NO: 99 inwhich the phenylalanine residue (Phe) at position 94 of SEQ ID NO: 99 isreplaced by a leucine residue (Leu).

Example 114 is the combination of HuVH1-HuIgG1 and the modified SEQ IDNO: 99 described above.

Example 115 is the combination of HuVH1-HuIgG2 and the modified SEQ IDNO: 99 described above.

Example 116 is the combination of HuVH1-HuIgG3 and the modified SEQ IDNO: 99 described above.

EXAMPLE 117-119

Expression of Humanised F(ab′)₂ Fragments with a Modified Light ChainVK4 Variable Sequence

The procedures described in Examples 39-47 are repeated with thevariable light chain sequence described in Example 109 used to make areplacement for the humanised light chain sequence of SEQ ID NO: 99 inwhich the tyrosine residue (Tyr) at position 57 and the phenylalanineresidue (Phe) at position 94 of SEQ ID NO: 99 is replaced by aphenylalanine residue (Phe) and leucine residue (Leu) respectively.

Example 117 is the combination of HuVH1-HuIgG1 and the modified SEQ IDNO: 99 described above.

Example 118 is the combination of HuVH1-HuIgG2 and the modified SEQ IDNO: 99 described above.

Example 119 is the combination of HuVH1-HuIgG3 and the 99 modified SEQID NO: described above.

EXAMPLE 120-122

Expression of Humanised F(ab′)₂ Fragments with a Chimeric Light ChainSequence

The procedures described in Examples 39-47 are repeated with thechimeric light chain sequence described in Example 110 replacing thehumanised light chain sequences used in Examples 39-47.

Example 120 is the combination of HuVH1-HuIgG1 and the chimeric lightchain sequence described above.

Example 121 is the combination of HuVH1-HuIgG2 and the chimeric lightchain sequence described above.

Example 122 is the combination of HuVH1-HuIgG3 and the chimeric lightchain sequence described above.

EXAMPLE 123

Preparation of Humanised Fusion Protein Based on Modified Light ChainVK4 Sequence

The procedures described in Example 48 are repeated but with plasmidpEE14-806.077HuVK4-HuCK replaced by a plasmid containing the modifiedVK4 sequence of Examples 107 and 111 to 113.

EXAMPLE 124

Preparation of Humanised Fusion Protein Based on Modified Light ChainVK4 Sequence

The procedures described in Example 48 are repeated but with plasmidpEE14-806.077HuVK4-HuCK replaced by a plasmid containing the modifiedVK4 sequence of Examples 108 and 114 to 116.

EXAMPLE 125

Preparation of Humanised Fusion Protein Based on Modified Light ChainVK4 Sequence

The procedures described in Example 48 are repeated but with plasmidpEE14-806.077HuVK4-HuCK replaced by a plasmid containing the modifiedVK4 sequence of Examples 109 and 117 to 119.

EXAMPLE 126

Preparation of Humanised Fusion Protein Based on a Chimeric Light ChainVK4 Sequence

The procedures described in Example 48 are repeated but with plasmidpEE14-806.077HuVK4-HuCK replaced by a plasmid containing the chimericlight chain sequence of Examples 110 and 120 to 122.

EXAMPLE 127

Preparation of Humanised Fusion Protein Based on Modified Light ChainVK4 Sequence

The procedures described in Example 75 are repeated but with plasmidpCF008/4 replaced by a plasmid containing the modified VK4 sequence ofExamples 107 and 111 to 113.

EXAMPLE 128

Preparation of Humanised Fusion Protein Based on Modified Light ChainVK4 Sequence

The procedures described in Example 75 are repeated but with plasmidpCF008/4 replaced by a plasmid containing the modified VK4 sequence ofExamples 108 and 114 to 116.

EXAMPLE 129

Preparation of Humanised Fusion Protein Based on Modified Light ChainVK4 Sequence

The procedures described in Example 75 are repeated but with plasmidpCF008/4 replaced by a plasmid containing the modified VK4 sequence ofExamples 109 and 117 to 119.

EXAMPLE 130

Preparation of Humanised Fusion Protein Based on a Chimeric Light ChainVK4 Sequence

The procedures described in Example 75 are repeated but with plasmidpCF008/4 replaced by a plasmid containing the chimeric light chainsequence of Examples 110 and 120 to 122.

REFERENCE EXAMPLE 1

Preparation of Gene Sequence for [G251T,D253K]HCPB

The method of cloning [G251T,D253K]HCPB in E.coli was very similar tothe method described in International Patent application Number WO96/2001 1, Example 15. Again pICI266 was used as the cloning vector, butthe starting material for PCR site directed mutagenesis was the[D253K]HCPB gene in plasmid p1CI1713 (as described in InternationalPatent Application Number WO 96/20011 Example 15). However, in this casesite directed mutagenesis was used during the PCR amplification of thegene to change the codon at amino acid position 251 in the mature genefrom Glycine to Threonine (GGC to ACT), the G251T change. Also duringthe generation of this mutation a number of other mutations weregenerated at the same (G251) site by using a mixture of oligonucleotideswith codon changes at G251. Individual mutant genes were identifiedfollowing transformation and hybridisation by sequencing across themutation site, prior to complete gene sequencing. In this example onlythe oligonucleotide for introducing the G251T mutation will beconsidered. Two PCR mixtures were prepared, in a manner similar to thatdescribed in International Patent application Number WO 96/20011 Example15. In the first reaction primers were CAN 00402 (SEQ ID NO: 116) andCAN 00734 (SEQ ID NO: 117). In the second reaction primers were CAN00284 (SEQ ID NO: 118) and CAN 01076 (SEQ ID NO: 119). In both reactionsthe starting DNA was pICI1713.

Aliquots of the two PCR reactions were analysed for DNA of the correctsize (about 750 and 250 base pairs) and estimation of concentration byagarose gel electrophoresis, and found to contain predominantly bands ofthe correct size. Another PCR was then set up using each of the firsttwo PCR products, with the two end primers {CAN 00402 (SEQ ID NO: 116)and CAN 00284 (SEQ ID NO: 118)}. An aliquot of the PCR product wasanalysed for DNA of the correct size (about 1000 base pairs) by agarosegel electrophoresis and found to contain predominantly a band of thecorrect size. The remainder of the product from the reaction mix waspurified, the isolated DNA restriction digested with enzymes NcoI andEcoRI, and a band of the correct size (about 1000 base pairs) purifiedin a similar manner to that described in international Patentapplication Number WO 96/20011 Example 16.

pICI266 double stranded DNA was restriction digested with NcoI and EcoRIenzymes, and DNA of the correct size (about 5600 base pairs) waspurified. Aliquots of both restricted and purified vector and insert DNAsamples were checked for purity and concentration estimation usingagarose gel electrophoresis compared with known standards. From theseestimates ligation mixes were prepared to clone the HCPB gene into thepICI266 vector in a similar manner to that described in InternationalPatent application Number WO 96/20011 Example 16.

Following the ligation reaction the DNA mixture was used to transformE.coli strain DH5α. colonies were picked and tested by hybridisation. Anumber of the clones were then taken for plasmid DNA preparation. andwere sequenced over the region of PCR mutation in order to identifyclones with the G251T change in a manner similar to that described inInternational Patent application Number WO 96/2001 1 Example 16. Fromthe sequencing results a clone containing a plasmid with the required[G25T:D253K]HCPB gene sequence was selected, and the plasmid calledpZEN1860.

REFERENCE EXAMPLE 2

Preparation of Gene Sequence for [A248S,G251T,D253K]HCPB

The method of cloning [A248S,G251T,D253K]HCPB in E.coli was very similarto the method described in Reference Example 1. The starting materialfor the PCR site directed mutagenesis was the [G251T,D253K]HCPB gene inplasmid pZEN1860 (described in Reference Example 1) in place ofpICI1713. However, in this case site directed mutagenesis was usedduring the PCR amplification of the gene to change the codon at aminoacid position 248 in the mature gene from alanine to serine (GCT toTTC), the A248S change. Two PCR mixtures were prepared, in a mannersimilar to that described in Reference Examples 1. In the first reactionprimers were CAN 00402 (SEQ ID NO: 116) and CAN 00720 (SEQ ID NO: 120).In the second reaction primers were CAN 00284 (SEQ ID NO: 118) and CAN00726 (SEQ ID NO: 121). In both reactions the starting DNA was pZEN1860.

Methods of PCR, cloning, expression and identification were the same asfor Reference Example 1. From the sequencing results a clone containinga plasmid with the required [A248S,G251T,D253K]HCPB gene sequence wasselected, and the plasmid called pZEN1921.

REFERENCE EXAMPLE 3

Preparation and Characterisation of a Human B7.1-Murine A5B7 F(ab′)₂Fusion Protein (AB7)

Methods for the preparation, purification and characterisation ofrecombinant murine A5B7 F(ab′)₂ antibody have been published (WO96120011, Reference Example 5). The cDNA sequence for human B7.1 antigen(also called CD80) has been isolated and described (Freeman G. J et al,Journal of Immunology, 1989, 143, 2714-2722). In this Example “A17”refers to human B7.1-murine A5B7 F(ab′)₂ fusion protein and “A5B7”refers to the anti-CEA antibody termed A5B7.

Using a PCR based strategy we isolated the natural signal sequence andextracellular domain of human B7.1 (encoding amino-acids 1-242) fromcDNA prepared from cultured Raji cells (ATCC No. CCL 86) and fused itdirectly upstream from the mature 5′ coding sequence of the murine A5B7Fd fragment. This involved isolation of the B7.1 sequence with PCRprimers 187/96 and 204/96 (SEQ ID NOS: 126 and 127) and a partial A5B7Fd sequence with PCR primers 203/96 and 205/96 (SEQ ID NOS: 128 and129). After purification of the PCR products they were mixed inapproximately equimolar amounts and fused by PCR with primers 187/96 and205/96. The resulting PCR product was purified, digested with HindIIIand BstEII (New England Biolabs (UK) Ltd., Wilbury Way, Hitchin, SG4OTY) and cloned into the HindIII-BstEII region of pAF1 using standardprocedures to create the full length human B7.1-murine A5B7 Fd fusion.This fusion gene (SEQ ID NO: 130-131) was cloned as a EcoRI-HindIIIfragment into the GS-system expression vector pEE6 (Celltech Biologics,Bath Road, Slough, SL1 4EN) according to the protocols described in WO96/20011, Reference Example 5, to generate vector pAB7.1.

A BglII-SalI fragment containing the B7.1-A5B7 Fd expression cassettewas then cloned between the BglII and SalI sites of the vector pAF6previously described to generate a vector (pAB7.2) capable ofco-expressing the fusion protein and the A5B7 L chain. The vector pAB7.2was then used to transform NSO myeloma cells and colonies selected ontheir ability to grow in the absence of glutamine. Cell lines expressingthe fusion protein were identified by determination of CEA bindingactivity in the culture supernatant using the ELISA described. A cellline expressing suitable levels of fusion protein (1D4) was selected forpurification and characterisation of the AB7 fusion protein.

Purification and Characterisation of the AB7Fusion Protein

The secreted recombinant B7.1(35-242)-A5B7 F(ab)₂, AB7, material waspurified from culture supernatant using a Protein-A agarose matrix suchas for example Protein-A Sepharose 4 fast flow as manufactured byPharmacia (Pharmacia Biotech, 23 Grosvenor Rd, St Albans, Herts, AL13AW). The matrix was washed with 2×8 matrix volumes of binding buffer(3M NaCl, 1.5M Glycine, pH 8.9). The culture supernatant containing AB7was diluted 1:1 with the binding buffer. The washed matrix was added tothe diluted culture supernatant (1 ml settled volume of matrix per 40 mlof diluted supernatant) and incubated at 4° C. for 2 hrs with moderateshaking. The matrix was spun down by centrifugation and approx. 75% ofthe supernatant carefully poured off. The matrix was then resuspended inthe residual supernatant and the resulting slurry packed into a column.The column was washed with 5-6 column volumes of 150 mM NaCl, 10 mMNaH₂PO₄, pH7.4. The buffer was then changed to 100 mM NaCitrate pH2.8and elution fractions collected. These fractions were titrated toapproximately pH7.0 by the addition of 2M Tris buffer pH.8.5. Theelution fractions were analysed by non-reducing SDS-PAGE and the peakAB7 fraction(s) retained as the product.

N-Terminal Sequencing

A sample of AB7 was run on reducing SDS-PAGE and blotted onto PVDF(polyvinylidene difluoride) membrane (equipment, gels, blotting membraneand methods from NOVEX, 4202 Sorrento Valley Blvd, San Diego, Calif.92121, USA.). The protein bands were stained with Coomassie blue and theband at approximately 70 kDa (i.e. B7.1-Fd fusion) was N-terminallysequenced (Applied Biosystems, 494 Protein Sequencer (Perkin Elmer, ABIdivision, Kelvin close, Birchwood Science Park North, Warrington, WA37PB.) The sequence obtained matched the expected sequence for mature B7(ie. after leader sequence cleavage from amino-acid 35 in SEQ.ID NO:131, Val Ile His Val etc.).

BIAcore Analysis

AB7 was analysed using BIAcore surface plasmon resonance equipment madeby Biacore (23 Grosvenor Rd, St. Albans, Herts., AL1 3AW, UK.) accordingto methods for BIAcore analysis of the CD80/CTLA-4 interaction takenfrom Greene J L, Leytze G M, Emswiler J, Peach R. Bajorath J, Cosand W,and Linsley P S. (1996) J. Biol. Chem. 271, 26762-26771. Samples of thepurified AB7 product were injected over both a CTLA4-Ig amine coupledsurface and a blank (control) amine coupled surface. Binding couldclearly be seen to the CTLA4-Ig surface compared to the control surface(see FIG. 3). Binding could also be demonstrated between CTLA4-Ig andAB7 when the CTLA4-Ig was injected over an amine coupled AB7 surface.

Combined with the data from the anti-CEA ELISA these data confirm thatthe purified AB7 fusion protein has the biological properties of bothcomponent parts, namely antigen and receptor binding activities.

Co-Stimulatory Activity of the AB7 Fusion Protein

The ability of the AB7 fusion protein to provide a co-stimulatory signalto T cells when bound to CEA expressing tumour cells was tested using anadaptation of a co-stimulation assay format previously described(Jenkins et al. (1991) J. Immunol. 147:2461). CEA expressing LS174Tcolo-rectal tumour cells (fixed using 0.5% paraformaldehyde for 5minutes at room temperature) were incubated with 10 μg/ml of the AB7fusion protein (2 hours rotating at 4° C. in RPMI 1640 medium (Gibco.Life Technologies, Paisley, Scotland), containing 0.5% human serum(Sigma AB, Sigma Chemical Co, Dorset, UK.). The cells were washed twiceprior to use and binding of the fusion protein confirmed using afluoroscein isothiocyanate (FITC)—conjugated goat-anti-mouse Ig(Becton-Dickinson UK Ltd, Oxford) and flow cytometry (Facscan, BectonDickinson). To allow the use of unprimed human T cells in the assay, theT cell receptor (TCR) stimulus was provided by an anti T cell receptorantibody (anti-CD3 antibody, OKT-3 Orthoclinical Diagnostics, Amersham,UK) previously coated onto the wells of a 96 well plate. OKT-3 wasimmobilised by incubating purified antibody (2 μg/ml in bicarbonatecoating buffer, pH 9.6 (preformed capsule, Sigma)) overnight at 4° C. in96 well flat bottomed microtitre plates (Costar Corporation, Cambridge,Mass., USA), which were then washed three to four times with PBS.Purified peripheral T cells (from negatively depleted (i.e. pullingout-components other than T cells) from donor human blood using magneticbeads (Dynabeads, Dynal A. S, Oslo, Norway) were added to the wells at2×10⁵/well in 50 μl of RPMI 1640 medium containing 5% human serum. Thefusion protein bound LS174T cells were added to the wells at 5×10⁴/wellin 50 μl of RPMI 1640 medium plus 5% human serum. Finally the volume inall wells was made up to 200 μl using RPMI 1640 medium plus 5% humanserum. Cultures were pulsed with 1.25 μCi of [³H] thymidine (AmershamInternational) after 48 hours and harvested 16 hours later with asemi-automated cell harvester (TomTec harvester, Wallac UK.). Theincorporation of [³H] thymidine into DNA was quantitated using liquidscintillation counting (Betaplate Scint and Betaplate counter, WallacUK.). Data from a typical costimulation assay is displayed in the Tablebelow.

TABLE Co-stimulation data αCD3 coated onto wells @ 2 μg/ml (cpm) T cellsalone  3582 T cells + αCD28 28178 T cells + LS174T 12303 T cells +LS174T + αCD28 25759 T cells + LS174T/fusion protein 41755 αCD3 =anti-CD3 antibody; αCD28 = anti-CD28 antibody

Unprimed T-cells require both T-cell receptor and co-stimulatorysignals. In the assay the T-cell receptor signal is provided by αCD3antibody. Providing co-stimulation via αCD28 (Becton-Dickinson used at0.6 μg/ml) stimulates uptake of [³H] thymidine over 8 fold compared toαCD3 alone. The presence of tumour cells has no significant effect onthis stimulation. Providing the co-stimulatory signal by AB7 fusionprotein bound to tumour cells stimulates uptake of [³H] thymidine bymore than 3 fold over that given by tumour cells alone and over 11 foldhigher than that seen in the absence of co-stimulation. The apparentstimulation provided by tumour cells alone may arise from residualaccessory cells in the purified T-cell population. Similar increases inT cell proliferation were consistently observed in wells containingtumour cell bound fusion protein in each of 5 assays carried outcompared with wells containing T cells and unbound tumour cells.

REFERENCE EXAMPLE 4

Preparation of IgG3-pBSIIKS+

This example describes the preparation of a vector containing a gene forthe human IgG3 heavy chain constant and hinge region.

A gene containing the sequence shown in SEQ ID NO: 115 [this contains asequence (residues 8 to 508) that is similar to SEQ ID NO: 25, but withresidues 312 and 501 of SEQ ID NO: 25 changed to C and G respectively],was prepared by PCR by a method similar to that described by Jayaramanet al. (1991) Proc. Natl. Acad. Sci USA 88, 4084-4088.

The gene was made in two parts, known as IgG3A and IgG3B. These werecloned separately into the SacI and XmaI sites of pBluescriptKS+(Stratagene Cloning Systems) to give vectors IgG3A-pBSIIKS+clone A7and IgG3B-pBSIIKS+clone B17 respectively. IgG3A was made to extend pastthe PmaCI restriction site (CACGTG at positions 334-339 in SEQ ID NO:115). Similarly, IgG3B was made such that the 5′ end of the sequence wasupstream of the PmaCI restriction site. To obtain the desired IgG3 genesequence, the intermediate IgG3A and IgG3B vectors were cut with AflIIIand PmaCI. The vector fragment (2823 bp) from IgG3A-pBSIIKS+clone A7,and insert fragment from IgG3B-pBSIIKS+clone B17 (666 bp) were isolatedby electrophoresis in a 1% agarose gel and purified. The fragments wereligated and the ligation mix used to transform E. coli strain DH5α.Clones containing the required gene were identified by digestion ofisolated DNA with SacI and XmaI to give a 520 bp fragment. The sequenceof the insert was confirmed by DNA sequence analysis and clone number F3was designated IgG3-pBSIIKS+.

What is claimed is:
 1. An anti-CEA (carcinoembryonic antigen) antibody(“806.077 Ab”) comprising complementarity determining regions (CDRs) inwhich the CDRs comprise the following sequences: a) heavy chain CDR1DNYMH (SEQ ID NO: 29) CDR2 WIDPENGDTE YAPKFRG (SEQ ID NO: 31) CDR3LIYAGYLAMD Y(SEQ ID NO: 32); and b) light chain CDR1 SASSSVTYMH (SEQ IDNO: 26) CDR2 STSNLAS (SEQ ID NO: 27) CDR3 QQRSTYPLT (SEQ ID NO: 28). 2.An antibody according to claim 1 in which the heavy chain CDRs 1 and 3are further defined as: CDR1 FNIKDNYMH (SEQ ID NO: 30); and CDR3HVLIYAGYLA MDY (SEQ ID NO: 33).
 3. An antibody according to claim 1comprising the following sequence: a heavy chain variable regionsequence (SEQ ID NO: 11) EVQLQQSGAE LVRSGASVKL SCTASGFNIK DNYMHWVKQR 40PEQGLEWIAW IDPENGDTEY APKFRGKATL TADSSSNTAY 80 LHLSSLTSED TAVYYCHVLIYAGYLAMDYW GQGTSVAVSS 120 and; a light chain variable region sequence(SEQ ID NO: 9): DIELTQSPAI MSASPGEKVT ITCSASSSVT YMHWFQQKPG 40TSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE 80 DAATYYCQQR STYPLTFGAGTKLELKRA
 108. 4. An antibody according to claim 3 which is in the formof a humanized antibody.
 5. A humanised antibody according to claim 4,comprising at least one of the following sequences: a heavy chainvariable region sequence which is VH1 (SEQ ID NO: 55); a light chainvariable region sequence which is VK4 (SEQ ID NO: 71); a human CH1 heavychain IgG3 constant region; a human kappa light chain CL region; and ahuman IgG3 hinge region.
 6. Hybridoma 806.077 deposited as ECACC depositno.
 96022936. 7. A method of making an antibody as defined in claim 1,2, 3, 5 or 4 which comprises: subjecting either a host cell transformedwith a polynucleotide sequence capable of encoding a polypeptide of anantibody defined in claim 1, 2, 3, 5 or 4 or the hybridoma of claim 6,to conditions conducive to expression.
 8. A humanized antibody accordingto claim 5 which is in the form of an F(ab′)₂ fragment.